Hydrazide containing nuclear transport modulators and uses thereof

ABSTRACT

The invention generally relates to nuclear transport modulators, e.g., CRM1 inhibitors, and more particularly to a compound represented by structural formula I: 
                         
or a pharmaceutically acceptable salt thereof, wherein the values and alternative values for the variables are as defined and described herein. The invention also includes the synthesis and use of a compound of structural formula I, or a pharmaceutically acceptable salt or composition thereof, e.g., in the treatment, modulation and/or prevention of physiological conditions associated with CRM1 activity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/235,306, which is the U.S. National Stage Application ofInternational Application No. PCT/US2012/048319, filed on Jul. 26, 2012,published in English, which claims the benefit of U.S. ProvisionalApplication No. 61/513,428, filed Jul. 29, 2011, U.S. ProvisionalApplication No. 61/513,432, filed Jul. 29, 2011, U.S. ProvisionalApplication No. 61/610,178, filed Mar. 13, 2012, U.S. ProvisionalApplication No. 61/654,651, filed Jun. 1, 2012, and U.S. ProvisionalApplication No. 61/653,588, filed May 31, 2012. The contents of theabove applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Cells from most major human solid and hematologic malignancies exhibitabnormal cellular localization of a variety of oncogenic proteins, tumorsuppressor proteins, and cell cycle regulators (Cronshaw et al. 2004,Falini et al 2006). For example, certain p53 mutations lead tolocalization in the cytoplasm rather than in the nucleus. This resultsin the loss of normal growth regulation, despite intact tumor suppressorfunction. In other tumors, wild-type p53 is sequestered in the cytoplasmor rapidly degraded, again leading to loss of its suppressor function.Restoration of appropriate nuclear localization of functional p53protein can normalize some properties of neoplastic cells (Cai et al.2008; Hoshino et al. 2008; Lain et al. 1999a; Lain et al. 1999b; Smartet al. 1999), can restore sensitivity of cancer cells to DNA damagingagents (Cai et al. 2008), and can lead to regression of establishedtumors (Sharpless & DePinho 2007, Xue et al. 2007). Similar data havebeen obtained for other tumor suppressor proteins such as forkhead(Turner and Sullivan 2008) and c-Abl (Vignari and Wang 2001). Inaddition, abnormal localization of several tumor suppressor and growthregulatory proteins may be involved in the pathogenesis of autoimmunediseases (Davis 2007, Nakahara 2009). CRM1 inhibition may provideparticularly interesting utility in familial cancer syndromes (e.g.,Li-Fraumeni Syndrome due to loss of one p53 allele, BRCA1 or 2 cancersyndromes), where specific tumor suppressor proteins (TSP) are deletedor dysfunctional and where increasing TSP levels by systemic (or local)administration of CRM1 inhibitors could help restore normal tumorsuppressor function.

Specific proteins and RNAs are carried into and out of the nucleus byspecialized transport molecules, which are classified as importins ifthey transport molecules into the nucleus, and exportins if theytransport molecules out of the nucleus (Terry et al. 2007; Sorokin etal. 2007). Proteins that are transported into or out of the nucleuscontain nuclear import/localization (NLS) or export (NES) sequences thatallow them to interact with the relevant transporters. ChromosomalRegion Maintenance 1 (Crm1 or CRM1), which is also called exportin-1 orXpo1, is a major exportin.

Overexpression of Crm1 has been reported in several tumors, includinghuman ovarian cancer (Noske et al. 2008), cervical cancer (van der Wattet al. 2009), pancreatic cancer (Huang et al. 2009), hepatocellularcarcinoma (Pascale et al. 2005) and osteosarcoma (Yao et al. 2009) andis independently correlated with poor clinical outcomes in these tumortypes.

Inhibition of Crm1 blocks the exodus of tumor suppressor proteins and/orgrowth regulators such as p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27,E2F4, KLF5, YAP1, ZAP, KLF5, HDAC4, HDAC5 or forkhead proteins (e.g.,FOXO3a) from the nucleus that are associated with gene expression, cellproliferation, angiogenesis and epigenetics. Crm1 inhibitors have beenshown to induce apoptosis in cancer cells even in the presence ofactivating oncogenic or growth stimulating signals, while sparing normal(untransformed) cells. Most studies of Crm1 inhibition have utilized thenatural product Crm1 inhibitor Leptomycin B (LMB). LMB itself is highlytoxic to neoplastic cells, but poorly tolerated with markedgastrointestinal toxicity in animals (Roberts et al. 1986) and humans(Newlands et al. 1996). Derivatization of LMB to improve drug-likeproperties leads to compounds that retain antitumor activity and arebetter tolerated in animal tumor models (Yang et al. 2007, Yang et al.2008, Mutka et al. 2009). Therefore, nuclear export inhibitors couldhave beneficial effects in neoplastic and other proliferative disorders.

In addition to tumor suppressor proteins, Crm1 also exports several keyproteins that are involved in many inflammatory processes. These includeIkB, NF-kB, Cox-2, RXRα, Commd1, HIF1, HMGB1, FOXO, FOXP and others. Thenuclear factor kappa B (NF-kB/rel) family of transcriptional activators,named for the discovery that it drives immunoglobulin kappa geneexpression, regulate the mRNA expression of variety of genes involved ininflammation, proliferation, immunity and cell survival. Under basalconditions, a protein inhibitor of NF-kB, called IkB, binds to NF-kB inthe nucleus and the complex IkB-NF-kB renders the NF-kB transcriptionalfunction inactive. In response to inflammatory stimuli, IkB dissociatesfrom the IkB-NF-kB complex, which releases NF-kB and unmasks its potenttranscriptional activity. Many signals that activate NF-kB do so bytargeting IkB for proteolysis (phosphorylation of IkB renders it“marked” for ubiquitination and then proteolysis). The nuclearIkBa-NF-kB complex can be exported to the cytoplasm by Crm1 where itdissociates and NF-kB can be reactivated. Ubiquitinated IkB may alsodissociate from the NF-kB complex, restoring NF-kB transcriptionalactivity. Inhibition of Crm1 induced export in human neutrophils andmacrophage like cells (U937) by LMB not only results in accumulation oftranscriptionally inactive, nuclear IkBa-NF-kB complex but also preventsthe initial activation of NF-kB even upon cell stimulation (Ghosh 2008,Huang 2000). In a different study, treatment with LMB inhibited IL-1βinduced NF-kB DNA binding (the first step in NF-kB transcriptionalactivation), IL-8 expression and intercellular adhesion moleculeexpression in pulmonary microvascular endothelial cells (Walsh 2008).COMMD1 is another nuclear inhibitor of both NF-kB and hypoxia-induciblefactor 1 (HIF1) transcriptional activity. Blocking the nuclear export ofCOMMD1 by inhibiting Crm1 results in increased inhibition of NF-kB andHIF1 transcriptional activity (Muller 2009).

Crm1 also mediates retinoid X receptor α (RXRα) transport. RXRα ishighly expressed in the liver and plays a central role in regulatingbile acid, cholesterol, fatty acid, steroid and xenobiotic metabolismand homeostasis. During liver inflammation, nuclear RXRα levels aresignificantly reduced, mainly due to inflammation-mediated nuclearexport of RXRα by Crm1. LMB is able to prevent IL-1β induced cytoplasmicincrease in RXRα levels in human liver derived cells (Zimmerman 2006).

The role of Crm1-mediated nuclear export in NF-kB, HIF-1 and RXRαsignalling suggests that blocking nuclear export can be potentiallybeneficial in many inflammatory processes across multiple tissues andorgans including the vasculature (vasculitis, arteritis, polymyalgiarheumatic, atherosclerosis), dermatologic (see below), rheumatologic(rheumatoid and related arthritis, psoriatic arthritis,spondyloarthropathies, crystal arthropathies, systemic lupuserythematosus, mixed connective tissue disease, myositis syndromes,dermatomyositis, inclusion body myositis, undifferentiated connectivetissue disease, Sjogren's syndrome, scleroderma and overlap syndromes,etc.).

CRM1 inhibition affects gene expression by inhibiting/activating aseries of transcription factors like ICp27, E2F4, KLF5, YAP1, and ZAP.

Crm1 inhibition has potential therapeutic effects across manydermatologic syndromes including inflammatory dermatoses (atopy,allergic dermatitis, chemical dermatitis, psoriasis), sun-damage(ultraviolet (UV) damage), and infections. CRM1 inhibition, best studiedwith LMB, showed minimal effects on normal keratinocytes, and exertedanti-inflammatory activity on keratinocytes subjected to UV, TNFα, orother inflammatory stimuli (Kobayashi & Shinkai 2005, Kannan & Jaiswal2006). Crm1 inhibition also upregulates NRF2 (nuclear factorerythroid-related factor 2) activity, which protects keratinocytes(Schafer et al. 2010, Kannan & Jaiswal 2006) and other cell types (Wanget al. 2009) from oxidative damage LMB induces apoptosis inkeratinocytes infected with oncogenic human papillomavirus (HPV) strainssuch as HPV16, but not in uninfected keratinocytes (Jolly et al. 2009).

Crm1 also mediates the transport of key neuroprotectant proteins thatmay be useful in neurodegenerative diseases including Parkinson'sdisease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis(ALS). For example, by (1) forcing nuclear retention of keyneuroprotective regulators such as NRF2 (Wang 2009), FOXA2 (Kittappa etal. 2007), parking in neuronal cells, and/or (2) inhibiting NFκBtranscriptional activity by sequestering IκB to the nucleus in glialcells, Crm1 inhibition could slow or prevent neuronal cell death foundin these disorders. There is also evidence linking abnormal glial cellproliferation to abnormalities in CRM1 levels or CRM1 function (Shen2008).

Intact nuclear export, primarily mediated through CRM1, is also requiredfor the intact maturation of many viruses. Viruses where nuclear export,and/or CRM1 itself, has been implicated in their lifecycle include humanimmunodeficiency virus (HIV), adenovirus, simian retrovirus type 1,Borna disease virus, influenza (usual strains as well as H1N1 and avianH5N1 strains), hepatitis B (HBV) and C(HCV) viruses, humanpapillomavirus (HPV), respiratory syncytial virus (RSV), Dungee, SevereAcute Respiratory Syndrome coronavirus, yellow fever virus, West Nilevirus, herpes simplex virus (HSV), cytomegalovirus (CMV), and Merkelcell polyomavirus (MCV). (Bhuvanakantham 2010, Cohen 2010, Whittaker1998). It is anticipated that additional viral infections reliant onintact nuclear export will be uncovered in the future.

The HIV-1 Rev protein, which traffics through nucleolus and shuttlesbetween the nucleus and cytoplasm, facilitates export of unspliced andsingly spliced HIV transcripts containing Rev Response Elements (RRE)RNA by the CRM1 export pathway. Inhibition of Rev-mediated RNA transportusing CRM1 inhibitors such as LMB or PKF050-638 can arrest the HIV-1transcriptional process, inhibit the production of new HIV-1 virions,and thereby reduce HIV-1 levels (Pollard 1998, Daelemans 2002).

Dengue virus (DENV) is the causative agent of the common arthropod-borneviral disease, Dengue fever (DF), and its more severe and potentiallydeadly Dengue hemorrhagic fever (DHF). DHF appears to be the result ofan over exuberant inflammatory response to DENV. NS5 is the largest andmost conserved protein of DENV. CRM1 regulates the transport of NS5 fromthe nucleus to the cytoplasm, where most of the NS5 functions aremediated. Inhibition of CRM1-mediated export of NS5 results in alteredkinetics of virus production and reduces induction of the inflammatorychemokine interleukin-8 (IL-8), presenting a new avenue for thetreatment of diseases caused by DENV and other medically importantflaviviruses including hepatitis C virus (Rawlinson 2009).

Other virus-encoded RNA-binding proteins that use CRM1 to exit thenucleus include the HSV type 1 tegument protein (VP13/14, or hUL47),human CMV protein pp 65, the SARS Coronavirus ORF 3b Protein, and theRSV matrix (M) protein (Williams 2008, Sanchez 2007, Freundt 2009,Ghildyal 2009).

Interestingly, many of these viruses are associated with specific typesof human cancer including hepatocellular carcinoma (HCC) due to chronicHBV or HCV infection, cervical cancer due to HPV, and Merkel cellcarcinoma associated with MCV. CRM1 inhibitors could therefore havebeneficial effects on both the viral infectious process as well as onthe process of neoplastic transformation due to these viruses.

CRM1 controls the nuclear localization and therefore activity ofmultiple DNA metabolizing enzymes including histone deacetylases (HDAC),histone acetyltransferases (HAT), and histone methyltransferases (HMT).Suppression of cardiomyocyte hypertrophy with irreversible CRM1inhibitors has been demonstrated and is believed to be linked to nuclearretention (and activation) of HDAC 5, an enzyme known to suppress ahypertrophic genetic program (Monovich et al. 2009). Thus, CRM1inhibition may have beneficial effects in hypertrophic syndromes,including certain forms of congestive heart failure and hypertrophiccardiomyopathies.

CRM1 has also been linked to other disorders. Leber's disorder, ahereditary disorder characterized by degeneration of retinal ganglioncells and visual loss, is associated with inaction of the CRM1 switch(Gupta N 2008). There is also evidence linking neurodegenerativedisorders to abnormalities in nuclear transport.

To date, however, small-molecule, drug-like Crm1 inhibitors for use invitro and in vivo are uncommon.

SUMMARY OF THE INVENTION

The present invention relates to compounds, or pharmaceuticallyacceptable salts thereof, useful as nuclear transport modulators. Theinvention also provides pharmaceutically acceptable compositionscomprising compounds of the present invention and methods of using saidcompounds and compositions in the treatment of various disorders, suchas those associated with abnormal cellular responses triggered byimproper nuclear transport.

In one embodiment of the invention, the compounds are represented byformula I:

or a pharmaceutically acceptable salt thereof, wherein the values andalternative values for each variable are as defined and describedherein.

Another embodiment of the invention is a composition comprising acompound of the invention, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

Yet another embodiment of the invention is a method for treating adisorder associated with CRM1 activity, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of the invention, or a pharmaceutically acceptablesalt thereof, or a composition comprising a compound of the invention,or a pharmaceutically acceptable salt thereof.

Another embodiment of the invention is use of a compound of theinvention for treating a disorder associated with CRM1 activity in asubject.

Another embodiment of the invention is use of a compound of theinvention for the manufacture of a medicament for treating a disorderassociated with CRM1 activity in a subject.

The nuclear transport modulators of the present invention, andpharmaceutically acceptable salts and/or compositions thereof, provideexcellent in vivo exposure as measured by AUC in mouse, rat, dog andmonkey, while exhibiting low levels of brain penetration. Therefore,compounds of the present invention, and pharmaceutically acceptablesalts and/or compositions thereof, are useful for treating a variety ofdiseases, disorders or conditions, associated with abnormal cellularresponses triggered by improper nuclear transport, such as thosediseases, disorders, or conditions described herein. Compounds providedby this invention are also useful for the study of nuclear transportmodulation in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by kinases; and thecomparative evaluation of nuclear transport modulators.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of tumor volume as a function of time and shows theeffect of Compound I-3 on tumor volume in a mouse xenograft model ofTriple Negative Breast Cancer (TNBC).

FIG. 2A is a Western blot image showing the effect of increasingconcentrations of Compound I-3 on CRM1 and apoptosis marker proteins inMDA-MB-468 TNBC cells.

FIG. 2B is a Western blot image showing the effect of increasingconcentrations of Compound I-3 on CRM1 and apoptosis marker proteins inDU4475 luminal BC cells.

FIG. 2C is a Western blot image showing the effect of increasingconcentrations of Compound I-3 on CRM1 and apoptosis marker proteins inHS578T TNBC cells.

FIG. 3 is Western blot images showing the effect of increasingconcentrations of Compound I-3 on anti-apoptosis and cell cycle proteinsin MDA-MB-468 and HS578T TNBC cell lines.

FIG. 4 is a graph of mean body weight versus time for days 0 to 12 inantibody-induced male BALB/c arthritic mice subjected to the indicatedtreatment.

FIG. 5 is a graph of mean total paw clinical arthritic scores versustime for days 0 to 12 in antibody-induced male BALB/c arthritic micesubjected to the indicated treatment.

FIG. 6 is a bar graph of scoring for mean ear thickness, scaling andfolding determined from day 0 to 7 in PMA-induced male BALB/c psoriaticmice subjected to the indicted treatment.

FIG. 7 is a set of graphs showing object preference of rats treated asindicted in the Novel Object Recognition Model.

FIG. 8A is a set of graphs showing cumulative and average food intakeversus time in obese and lean Zucker rats treated as indicated.

FIG. 8B is a set of graphs showing average and percent body weightversus time in obese and lean Zucker rats treated as indicated.

DETAILED DESCRIPTION

The novel features of the present invention will become apparent tothose of skill in the art upon examination of the following detaileddescription of the invention. It should be understood, however, that thedetailed description of the invention and the specific examplespresented, while indicating certain embodiments of the presentinvention, are provided for illustration purposes only because variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those of skill in the art from the detaileddescription of the invention and claims that follow.

Compounds of the Invention

One embodiment of the invention is compounds represented by formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from hydrogen and methyl;

R² is selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,pyrazin-2-yl, and quinoxalin-2-yl, pyrimidin-4-yl,1,1-dioxotetrahydrothiophen-3-yl and cyclopropyl, wherein R² isoptionally substituted with one or more independent substituentsselected from methyl and halogen; or

R¹ and R² are taken together with their intervening atoms to form4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl,4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3-hydroxyazetidin-1-yl,or morpholin-4-yl;

R³ is selected from hydrogen and halo; and

represents a single bond wherein a carbon-carbon double bond boundthereto is in an (E)- or (Z)-configuration.

As described generally above, R¹ is selected from hydrogen and methyl.In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is methyl.

As described generally above, R² is selected from pyridin-2-yl,pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, quinoxalin-2-yl,pyrimidin-4-yl, 1,1-dioxotetrahydrothiophen-3-yl and cyclopropyl,wherein R² is optionally substituted with one or more independentsubstituents selected from methyl and halogen. In some embodiments offormula I, R² is pyridin-2-yl. In some embodiments of formula I, R² ispyridin-3-yl. In some embodiments of formula I, R² is pyridin-4-yl. Insome embodiments of formula I, R² is pyrazin-2-yl. In some embodimentsof formula I, R² is pyrimidin-4-yl. In some embodiments of formula I, R²is quinoxalin-2-yl. In some embodiments of formula I, R² is selectedfrom pyridin-2-yl, pyridin-3-yl and pyridin-4-yl. In some embodiments offormula I, R² is selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,pyrazin-2-yl and pyrimidin-4-yl. In some embodiments of formula I, R² isselected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl andpyrimidin-4-yl.

In some embodiments, R² is selected from:

In some embodiments of formula I, R² is optionally substituted with asingle substituent selected from methyl and chloro. In some embodimentsof formula I, R² is optionally substituted with a methyl group. In someembodiments of formula I, R² is optionally substituted with a chlorogroup. In some embodiments, R² is selected from:

In some embodiments, R² is selected from:

In some embodiments, R² is selected from:

In some embodiments of formula I, R¹ and R² are taken together withtheir intervening atoms to form 4-hydroxypiperidin-1-yl,pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl,4-ethylpiperazin-1-yl, 3-hydroxyazetidin-1-yl, or morpholin-4-yl. Insome embodiments of formula I, R¹ and R² are taken together with theirintervening atoms to form 4-hydroxypiperidin-1-yl.

As described generally above, R³ is selected from hydrogen and halogen.In some embodiments, R³ is hydrogen. In some embodiments, R³ is halogen(e.g., chloro, bromo, iodo or fluoro). In some such embodiments, R³ ischloro.

As described generally above, the carbon-carbon double bond in betweenthe triazole moiety and the carbonyl moiety is in an (E)-configurationor a (Z)-configuration. In some embodiments, that double bond is in a(E)-configuration. In some embodiments, that double bond is in a(Z)-configuration and the compound is represented by formula II:

or a pharmaceutically acceptable salt thereof, wherein R¹, R² and R³ areas defined above and described herein.

A further embodiment of the invention is a compound represented byformula II, or a pharmaceutically acceptable salt thereof, wherein thevalues and alternative values for the variables are as defined above fora compound of formula I.

In a first aspect of this further embodiment, R¹ is as defined above;and R² is selected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl andpyrimidin-4-yl, wherein R² is optionally substituted with a singlesubstituent selected from methyl and chloro; or R¹ and R² are takentogether with their intervening atoms to form 4-hydroxypiperidin-1-yl.

In a specific aspect of the first aspect R³ is hydrogen. The values andalternative values for the remaining variables are as described abovefor a compound of formula I, or in the further embodiment, or firstaspect thereof.

Exemplary compounds of formula I are set forth in Table 1.

TABLE 1 Exemplary compound of formula I.

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

In some embodiments, the compound of the invention is selected from anyone of compounds I-3 to I-26. In one aspect of these embodiments, thecompound is selected from compounds I-3, I-4, I-5, I-7, I-8, I-10, I-12,I-18, I-19 and I-24. In a more specific aspect, the compound of theinvention is selected from I-3 and I-4.

Pharmacokinetics (PK) play an increasing role in drug discovery anddevelopment. Pharmacokinetics is the quantitative study of the timecourse of drug absorption, distribution, metabolism and/or excretion.When a drug is administered, it distributes rapidly from itsadministration site into the systemic blood circulation. One measure ofthe extent of a therapeutic agent's distribution is the area under theplasma concentration-time curve (AUC), calculated to the last measuredconcentration (AUC_(t)) and extrapolated to infinity (AUC_(Inf)). AUC isthus a useful metric to quantitate drug exposure.

Generally, the higher the exposure of a therapeutic agent, the greaterthe effects of the agent. However, high exposure of a therapeutic agentmay have deleterious effects on certain tissues such as the brain. Whilethe blood-brain barrier (BBB), a protective network consisting of tightjunctions between endothelial cells, restricts the diffusion ofhydrophilic and/or large molecules, drugs with high AUC are stillcapable of penetrating the BBB and/or cerebrospinal fluid. Suchpenetration is often undesirable and can lead to unwanted side effects.Current drug discovery efforts are aimed, in part, at striking a balancebetween maximizing drug exposure (e.g., AUC), while minimizing brainpenetration.

The brain to plasma (B:P) ratio is one method of quantifying therelative distribution of a therapeutic agent in brain tissue to that incirculation and, as such, provides one indication of the brainpenetration of a given therapeutic agent. A high brain to plasma ratiois preferred when targeting diseases localized in the central nervoussystem (CNS), including the brain and the cerebrospinal fluid. However,a lower brain to plasma ratio is generally preferable for non-CNStherapeutic agents to minimize brain penetration and avoid potentialside effects caused by unwanted accumulation of the therapeutic agentsin the brain and CNS tissue.

As set forth in more detail in the Exemplification, the compounds of thepresent invention display a higher AUC and/or a lower B:P as compared toother nuclear transport inhibitors, such as those disclosed in co-ownedU.S. patent application Ser. No. 13/041,377, filed Mar. 5, 2011 andpublished as US 2009/0275607 on Nov. 10, 2011. In some embodiments ofthe present invention, the compound of formula I has a nuclear exportactivity of less than about 1 μM, an AUC_(Inf) of greater than about3300 (e.g., greater than about 3500), and a B:P ratio of less than about2.5 when dosed in a mouse at 10 mg/kg po.

Synthetic Methods of the Invention

In accordance with the present invention, there is provided a method ofpreparing (Z)-olefin derivatives of a compound of formula Z useful inpreparing compound of the invention (e.g., precursors to the compoundsof the invention):

or a pharmaceutically acceptable salt thereof, wherein:

Ring A is an optionally substituted ring selected from phenyl, an8-10-membered bicyclic aryl ring, a 5-6-membered monocyclic heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, and an 8-10 membered bicyclic heteroaryl ring having1-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur;

Y is a covalent bond or -L-;

L is a bivalent C₁₋₈ saturated or unsaturated, straight or branched,hydrocarbon radical, wherein one or two methylene units of L isoptionally replaced by —NR—, —N(R)C(O)—, —C(O)N(R)—, —O—, —C(O)—,—OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(S)—, —C(NOR)— or —C(NR)—;

each R is independently hydrogen or an optionally substituted groupselected from C₁₋₆ aliphatic, phenyl, a 4-7-membered saturated orpartially unsaturated carbocyclic ring, a 4-7-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5-6-memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, an 8-10-membered bicyclic aryl ring,and an 8-10-membered bicyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur; or

two R groups on the same nitrogen are taken together with the nitrogenatom to which they are attached to form a 4-7-membered saturated orpartially unsaturated heterocyclic ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or a5-6-membered heteroaryl ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur;

each of V¹, V² and V³ is independently C(R^(y)) or N;

each R^(x) and R^(y) is independently selected from —R, halogen, —OR,—SR, —N(R)₂, —CN, —NO₂, —N₃, —SOR, —SO₂R, —SO₂NR, —C(O)R, —CO₂R,—C(O)OR, —C(O)N(R)₂, —NRC(O)R, —OC(O)R, —OC(O)N(R)₂, —NRC(O)OR,—NRC(O)NR₂ and —NRSO₂R;

each R¹ and R² is independently hydrogen, deuterium, tritium or halogen;

W is —CN, haloalkyl, —NO₂ or —C(═Z)R³;

Z is O, S, or NR;

R³ is selected from hydrogen, —R, OR, —SR and —N(R⁴)₂;

each R⁴ is independently —R; or

two R⁴ on the same nitrogen are taken together with the nitrogen atom towhich they are attached to form a 4-7-membered saturated or partiallyunsaturated heterocyclic ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, or a 5-6-membered heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, wherein the ring thereby formed is optionallysubstituted with —(R⁵)_(n);

each R⁵ is independently selected from —R, halogen, —OR, —SR, —N(R)₂,—CN, —NO₂, —N₃, —SOR, —SO₂R, —SO₂NR, —C(O)R, —CO₂R, —C(O)OR, —C(O)N(R)₂,—NRC(O)R, —OC(O)R, —OC(O)N(R)₂, —NRC(O)OR, —NRC(O)NR₂ and —NRSO₂R; and

each m and n is independently an integer selected from 0, 1, 2, 3 and 4.

Compounds of formula Z have been described, for example, in U.S. Ser.No. 13/041,377, filed Mar. 5, 2011, and in U.S. Provisional ApplicationNos. 61/513,428, filed Jul. 29, 2011, and 61/653,588, filed Jun. 1,2012. Compounds of formula Z are generally synthesized as a mixture of(E)- and (Z)-olefin isomers, which must be separated. The separation of(E)- and (Z)-olefin isomers requires extensive chromatography andresults in a loss of 50% of the advanced intermediate A, as theundesired isomer cannot typically be converted to the desired isomer. A50% yield is inefficient and costly at any step of a synthesis, but suchunacceptable yields are even more problematic at the end of a multi-stepsynthesis. It has now been surprisingly discovered that the use ofsterically hindered bases in a 1,4-nucleophilic addition can effect(Z)-selectivity of the reaction, thereby providing the cis-olefin isomeras the major or exclusive product. Accordingly, the present inventionprovides a (Z)-selective synthesis of compounds of formula Z, andmethods of preparing synthetic intermediates useful for preparingcompounds of formula Z. A key step in the synthesis of compounds offormula Z is depicted in Scheme I.

In certain embodiments, the compounds of formula Z are preparedaccording to Scheme I, set forth below:

wherein LG is a leaving group and each of Ring A, Y, V¹, V², V³, R^(x),R¹, R², W and m is as defined above with respect to a compound offormula Z and described in embodiments herein.

In some embodiments of step S-1.1, intermediate A is coupled withintermediate B via a 1,4-nucleophilic addition/elimination reaction. Insome embodiments of step S-1.1, LG is a suitable leaving group. In somesuch embodiments of step S-1.1, LG is a halogen. In some embodiments, LGis iodo. In some embodiments of step S-1.1, LG is bromo. In someembodiments of step S-1.1, LG is a sulfonate. In some such embodiments,LG is methanesulfonate (mesylate).

In some embodiments of step S-1.1, intermediate A is coupled withintermediate B in the presence of a sterically-hindered nucleophilicbase. One of ordinary skill will be able to select a suitablesterically-hindered base. Suitable sterically-hindered nucleophilicbases for use in the present invention include1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane(DABCO), N,N-dicyclohexylmethylamine,2,6-di-tert-butyl-4-methylpyridine, quinuclidine,1,2,2,6,6-pentamethylpiperidine (PMP),7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine,tri-tert-butylphosphine and tricyclohexylphosphine.

In certain embodiments, the compounds of formula Y are preparedaccording to Scheme II, set forth below:

wherein LG is a leaving group and each of R^(x), R^(y), R¹, R², W and mis as defined above with respect to a compound of formula Z anddescribed in embodiments herein.

In some embodiments of step S-2.1, intermediate C is reacted with athiolate salt to provide intermediate D. In some embodiments of stepS-2.1, the thiolate salt is sodium thiolate. In some embodiments of stepS-2.1, the thiolate salt is potassium thiolate.

At step S-2.2, intermediate D is reacted with a hydrazine equivalent toprovide intermediate E.

At step S-2.3, intermediate E is coupled with intermediate B to providea compound of formula Y. In some embodiments of step S-2.3, LG is asuitable leaving group. In some such embodiments of step S-2.3, LG is ahalogen. In some embodiments, LG is iodo. In some embodiments of stepS-2.3, LG is bromo. In some embodiments of step S-2.3, LG is asulfonate. In some such embodiments, LG is methanesulfonate (mesylate).

In some embodiments of step S-2.3, intermediate E is coupled withintermediate B in the presence of a sterically-hindered nucleophilicbase. One of ordinary skill will be able to select a suitablesterically-hindered base. Suitable sterically-hindered nucleophilicbases for use in the present invention include1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane(DABCO), N,N-dicyclohexylmethylamine,2,6-di-tert-butyl-4-methylpyridine, quinuclidine,1,2,2,6,6-pentamethylpiperidine (PMP),7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine,tri-tert-butylphosphine and tricyclohexylphosphine.

According to one aspect, the present invention provides a method forproviding a compound of formula Z:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,Y, V¹, V², V³, R^(x), R, R¹, R², W and m is as defined above withrespect to a compound of formula Z,

comprising the steps of:

(a) providing a compound of formula A:

wherein each of Ring A, R^(x), Y, V¹, V², V³ and m is as defined abovefor a compound of formula Z; and

(b) reacting said compound of formula A with an olefin of formula B:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, W, R¹ and R² is as defined above for a compound of formula Z;

in the presence of a sterically-hindered nucleophilic base to form acompound of formula Z.

As described above, a compound of formula A is coupled with intermediateB via a 1,4-nucleophilic addition/elimination reaction. In someembodiments, a compound of formula A is coupled with intermediate B inthe presence of a sterically-hindered nucleophilic base. Suitablesterically-hindered bases include tertiary amine bases. In someembodiments, a suitable sterically-hindered bases includessterically-hindered secondary amine bases. In some embodiments, thesterically-hindered nucleophilic base is selected from1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane(DABCO), N,N-dicyclohexylmethylamine,2,6-di-tert-butyl-4-methylpyridine, quinuclidine,1,2,2,6,6-pentamethylpiperidine (PMP),7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine,tri-tert-butylphosphine and tricyclohexylphosphine. In some embodiments,the sterically-hindered nucleophilic base is1,4-diazabicyclo(2.2.2)octane (DABCO). In some embodiments, thesterically-hindered nucleophilic base is1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the sterically-hindered nucleophilic base is aphosphine. In some such embodiments, the sterically-hinderednucleophilic base is triphenylphosphine.

In some embodiments, step (b) above is performed at a temperature rangeof about 0° C. to about 100° C. In some embodiments, step (b) isperformed at a temperature of about 0° C. In some embodiments, step (b)is performed at a temperature of about 25° C. In some embodiments, step(b) is performed at a temperature of about 50° C. In some embodiments,step (b) is performed at a temperature of about 100° C.

One of ordinary skill will recognize that the 1,4-nucleophilicaddition/elimination reaction of a compound of formula A andintermediate B requires the use of a polar, aprotic organic solvent.Suitable polar, aprotic organic solvents include ethers such as dioxane,tetrahydrofuran and methyl tert-butyl ether (MTBE), and amides such asdimethylformamide (DMF) and dimethylacetamide (DMA). One of ordinaryskill is capable of selecting the appropriate solvent for the desiredreaction temperature.

According to another aspect, the present invention provides a method ofproviding a compound of formula Y:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R², W and m is as defined above with respect to a compound offormula Z,

comprising the steps of:

(a) providing a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula Y; and

(b) reacting said compound of formula E with an olefin of formula B:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, W, R¹ and R² is as defined above for a compound of formula Y,

in the presence of a sterically-hindered nucleophilic base to form acompound of formula Y.

As described above, a compound of formula E is coupled with intermediateB via a 1,4-nucleophilic addition/elimination reaction. In someembodiments, a compound of formula E is coupled with intermediate B inthe presence of a sterically-hindered nucleophilic base. Suitablesterically-hindered bases include tertiary amine bases. In someembodiments, a suitable sterically-hindered bases includessterically-hindered secondary amine bases. In some embodiments, thesterically-hindered nucleophilic base is selected from1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane(DABCO), N,N-dicyclohexylmethylamine,2,6-di-tert-butyl-4-methylpyridine, quinuclidine,1,2,2,6,6-pentamethylpiperidine (PMP),7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine,tri-tert-butylphosphine and tricyclohexylphosphine. In some embodiments,the sterically-hindered nucleophilic base is1,4-diazabicyclo(2.2.2)octane (DABCO). In some embodiments, thesterically-hindered nucleophilic base is1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the sterically-hindered nucleophilic base is aphosphine. In some such embodiments, the sterically-hinderednucleophilic base is triphenylphosphine.

In some embodiments, step (b) above is performed at a temperature rangeof about 0° C. to about 100° C. In some embodiments, step (b) isperformed at a temperature of about 0° C. In some embodiments, step (b)is performed at a temperature of about 25° C. In some embodiments, step(b) is performed at a temperature of about 50° C. In some embodiments,step (b) is performed at a temperature of about 100° C.

One of ordinary skill will recognize that the 1,4-nucleophilicaddition/elimination reaction of a compound of formula E andintermediate B requires the use of a polar, aprotic organic solvent.Suitable polar, aprotic organic solvents include ethers such as dioxane,tetrahydrofuran and methyl tert-butyl ether (MTBE), and amides such asdimethylformamide (DMF) and dimethylacetamide (DMA). One of ordinaryskill is capable of selecting the appropriate solvent for the desiredreaction temperature.

In some embodiments of a compound of formula Y, W is —CN. In someembodiments, W is haloalkyl. In some such embodiments, W is —CF₃. Insome embodiments, W is —NO₂.

In some embodiments, W is —C(═Z)R³. In some such embodiments, Z is O. Insome embodiments, W is —C(O)R³, wherein R³ is selected from —OR, —SR or—N(R⁴)₂. In some embodiments, W is —C(O)OR. In some embodiments, W is—C(O)OR, wherein R is selected from methyl, ethyl, isopropyl, butyl,tert-butyl and sec-butyl. In some embodiments, W is —C(O)OCH₃. In someembodiments, W is —C(O)OCH₂CH₃. In some embodiments, W is—C(O)OCH(CH₃)₂.

In some embodiments, W is —C(O)N(R⁴)₂. In some embodiments, W is—(O)NH(R⁴). In some embodiments, W is —C(O)NH₂. In some embodiments, Wis —C(═O)N(R⁴)₂, wherein both R⁴ groups are taken together with thenitrogen atom to which they are attached to form a 4-7 memberedsaturated heterocyclic ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, wherein the ring therebyformed is optionally substituted with —(R⁵)_(n). In some embodiments, Wis —C(O)N(R⁴)₂, wherein both R⁴ groups are taken together with thenitrogen atom to which they are attached to form a 4-7 memberedsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, wherein the ring therebyformed is optionally substituted with —(R⁵)_(n). In some embodiments, Wis —C(O)N(R⁴)₂, wherein both R⁴ groups are taken together with thenitrogen atom to which they are attached to form a 4-7 memberedsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein the ring therebyformed is optionally substituted with —(R⁵)_(n). In some embodiments, Wis —C(O)N(R⁴)₂, wherein both R⁴ groups are taken together with thenitrogen atom to which they are attached to form a 4-7-memberedsaturated heterocyclic ring having 1 nitrogen atom, wherein the ringthereby formed is optionally substituted with —(R⁵)_(n).

In some embodiments, W is —C(O)N(R⁴)₂, wherein both R⁴ groups are takentogether with the nitrogen atom to which they are attached to form a4-6-membered saturated heterocyclic ring having 1 nitrogen atom, whereinthe ring thereby formed is optionally substituted with —(R⁵)_(n). Insome embodiments, W is —C(O)N(R⁴)₂, wherein both R⁴ groups are takentogether with the nitrogen atom to which they are attached to form a4-5-membered saturated heterocyclic ring having 1 nitrogen atom, whereinthe ring thereby formed is optionally substituted with —(R⁵)_(n). Insome embodiments, W is —C(O)N(R⁴)₂, wherein both R⁴ groups are takentogether with the nitrogen atom to which they are attached to form a4-membered saturated heterocyclic ring having 1 nitrogen atom, whereinthe ring thereby formed is optionally substituted with —(R⁵)_(n). Insome embodiments, W is —C(O)N(R⁴)₂, wherein both R⁴ groups are takentogether with the nitrogen atom to which they are attached to form a4-membered saturated heterocyclic ring having 1 nitrogen atom, whereinthe ring thereby formed is substituted with at least one fluorine. Insome embodiments, W is —C(O)N(R⁴)₂, wherein both R⁴ groups are takentogether with the nitrogen atom to which they are attached to form a4-membered saturated heterocyclic ring having 1 nitrogen atom, whereinthe ring thereby formed is substituted with at least two fluorines. Insome embodiments, W is

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ isdeuterium.

In some embodiments, R² is hydrogen. In some embodiments, R² isdeuterium. In some embodiments, R¹ and R² are each hydrogen.

In some embodiments, m is 1. In some embodiments, m is 2. In some suchembodiments, R^(x) is haloalkyl. In some embodiments, R^(x) is —CF₃.

In some embodiments, R^(y) is hydrogen.

In some embodiments, the present invention provides a method ofproviding a compound of formula E:

wherein R^(x), R^(y) and m are as described for a compound of formula Z,

comprising the steps of:

(a) providing a compound of formula D:

wherein each of R^(x) and m is as defined above for a compound offormula E; and

(b) reacting said compound of formula D to form a compound of formula E.

In some embodiments, conditions effective to form a compound of formulaD includes a hydrazine equivalent. Thus, in some embodiments, step (b)of the method of providing a compound of formula E includes reactionsaid compound of formula D with a hydrazine equivalent to the form thecompound of formula E. In some embodiments, intermediate D is reactedwith hydrazine hydrate to provide a compound of formula E. In someembodiments, intermediate D is reacted with a protected form ofhydrazine such as tert-butyl hydrazinecarboxylate and subsequentlydeprotected to provide intermediate D.

One of ordinary skill will recognize that the addition of hydrazine tointermediate D requires a polar, aprotic organic solvent. Suitablepolar, aprotic organic solvents include ethers such as dioxane,tetrahydrofuran and methyl tert-butyl ether (MTBE), alcohols such asisopropyl alcohol, and amides such as dimethylformamide (DMF) anddimethylacetamide (DMA). One of ordinary skill is capable of selectingthe appropriate solvent for the desired reaction temperature.

In some embodiments, the present invention provides a method forpreparing a compound of formula D:

wherein R^(x) and m are as defined above for a compound of formula Z,

comprising the steps of:

(a) providing a compound of formula C:

wherein each of R^(x) and m is as defined above for a compound offormula D; and

(b) reacting said compound of formula C to form a compound of formula D.

As described above, in some embodiments, intermediate C is treated witha thiolate salt to provide intermediate D. In some embodiments, thethiolate salt is sodium thiolate. One of ordinary skill will recognizethat the reaction of intermediate C with a thiolate salt requires theuse of a polar, aprotic solvent. Suitable polar, aprotic solventsinclude ethers such as dioxane, tetrahydrofuran and methyl tert-butylether (MTBE).

In some embodiments, the present invention provides a method forpreparing a compound of formula B:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹, R² and W are as defined above for a compound of formulaZ,

comprising the steps of:

(a) providing a compound of formula F:

wherein each of R² and W is as defined above for a compound of formulaB; and

(b) reacting said compound of formula F to form a compound of formula B.

As described above, in some embodiments of intermediate B, LG is ahalogen. In some such embodiments, a compound of formula F is treatedwith a halide salt. In some embodiments, a compound of formula F istreated with a sodium halide. In some such embodiments, a compound offormula F is treated with sodium iodide. In some embodiments,intermediate F is treated with a halide salt in the presence of an acid.Suitable acids include both mineral acids and organic acids. In someembodiments, intermediate F is treated with a halide salt and an organicacid such as acetic acid. In some embodiments, intermediate F is treatedwith sodium iodide in the presence of acetic acid to provide a compoundof formula B.

One of ordinary skill will recognize that the addition of a halide saltto intermediate F requires a polar, aprotic organic solvent. Suitablepolar, aprotic organic solvents include ethers such as dioxane,tetrahydrofuran and methyl tert-butyl ether (MTBE).

According to another aspect, the present invention provides a method ofproviding a compound of formula X:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R², R⁴ and m is as defined above with respect to a compoundof formula Z,

comprising the steps of:

(a) providing a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula X; and

(b) reacting said compound of formula E with an olefin of formula G:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹, R² and R⁴ is as defined above for a compound of formulaX,

in the presence of a sterically-hindered nucleophilic base to form acompound of formula X.

According to another aspect, the present invention provides a method ofproviding a compound of formula W:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R², R⁵, m and n is as defined above with respect to acompound of formula Z,

comprising the steps of:

(a) providing a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula W; and

(b) reacting said compound of formula E with an olefin of formula H:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹, R², R⁵ and n is as defined above for a compound offormula W,

in the presence of a sterically-hindered nucleophilic base to form acompound of formula W.

According to another aspect, the present invention provides a method ofproviding a compound of formula V:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R², and m is as defined above with respect to a compound offormula Z,

comprising the steps of:

(a) providing a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula V; and

(b) reacting said compound of formula E with an olefin of formula J:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹ and R² is as defined above for a compound of formula V,

in the presence of a sterically-hindered nucleophilic base to form acompound of formula V.

In some embodiments, the present invention provides a method forpreparing a compound of formula G:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹, R² and R⁴ is as described herein with respect to acompound of formula Z,

comprising the steps of:

(a) providing a compound of formula K:

wherein each of R² and R⁴ is as defined above for a compound of formulaG; and

(b) reacting said compound of formula K to form a compound of formula G.

As described above, in some embodiments of intermediate G, LG is ahalogen. In some such embodiments, a compound of formula K is treatedwith a halide salt. In some embodiments, a compound of formula K istreated with a sodium halide. In some such embodiments, a compound offormula K is treated with sodium iodide. In some embodiments,intermediate K is treated with a halide salt in the presence of an acid.Suitable acids include both mineral acids and organic acids. In someembodiments, intermediate K is treated with a halide salt and an organicacid such as acetic acid. In some embodiments, intermediate K is treatedwith sodium iodide in the presence of acetic acid to provide a compoundof formula G.

In some embodiments, the present invention provides a method forpreparing a compound of formula K:

wherein each of R² and R⁴ is as defined above with respect to a compoundof formula Z,

comprising the steps of:

(a) providing a compound of formula L:

wherein R² is hydrogen, deuterium, tritium or halogen; and

(b) reacting said compound of formula L with HN(R⁴)₂, wherein each R⁴ isas defined above with respect to a compound of formula K, to form acompound of formula K.

In some embodiments, a compound of formula L is treated with an amidecoupling agent in the presence of HN(R⁴)₂ to form a compound of formulaK. Suitable amide coupling agents include HOBt, HOAt, HAMDU, HAMTU,PyBOP, PyBrOP, TBTU, HATU and T3P. One of ordinary skill will recognizethat the use of such amide coupling reagents requires the use of a base.Suitable bases include organic bases, such as triethylamine,diisopropylethyl amine, pyridine, 4-dimethylpyridine (DMAP), and thelike.

In some embodiments, a compound of formula L is reacted with achlorinating agent such as thionyl chloride to form an acyl chloride,which is then reacted with HN(R⁴)₂ to form a compound of formula K.

In some embodiments, the present invention provides a method forpreparing a compound of formula G:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹, R² and R⁴ is as defined above with respect to a compoundof formula Z,

comprising the steps of:

(a) providing a propargylic acid of formula L:

wherein R² is as defined above for a compound of formula G;

(b) reacting said compound of formula L with an alcohol having theformula HO—R to form a propargylic ester of formula M:

wherein each of R and R² is as defined above for a compound of formulaG;

(c) reacting said propargylic ester of formula M to form a compound offormula N:

wherein each of R, R¹, R² and LG is as defined above for a compound offormula G;

(d) hydrolyzing said compound of formula N to form a compound of formulaQ:

wherein each of R, R¹, R² and LG is as defined above for a compound offormula G; and

(e) reacting said compound of formula Q with HN(R⁴)₂, wherein each R⁴ isas defined above for a compound of formula G, to form a compound offormula G.

In some embodiments, a propargylic acid of formula L is treated with analcohol to form a propargylic ester of formula M. Suitable alcoholsinclude methanol, ethanol and isopropanol. One of ordinary skill willrecognize that the esterification of a propargylic acid of formula L canbe effected by catalytic acid. Thus, in some embodiments, a propargylicacid of formula L is treated with methanol or ethanol in the presence ofcatalytic sulfuric acid to provide a propargylic ester of formula M.

One of ordinary skill will recognize that such esterification can beperformed at temperatures of about 25° C. to about 100° C., or up to theboiling point of the alcohol. In some embodiments, the esterification ofa propargylic acid of formula L is heated to reflux (the boiling pointof the alcohol).

As described above, in some embodiments of a compound of formula N, LGis a halogen. In some such embodiments, a compound of formula M istreated with a halide salt. In some embodiments, a compound of formula Mis treated with a sodium halide. In some such embodiments, a compound offormula M is treated with sodium iodide. In some embodiments, a compoundof formula M is treated with a halide salt in the presence of an acid.Suitable acids include both mineral acids and organic acids. In someembodiments, a compound of formula M is treated with a halide salt andan organic acid such as acetic acid. In some embodiments, a compound offormula M is treated with sodium iodide in the presence of acetic acidto provide a compound of formula N.

In some embodiments, the ester of a compound of formula N is hydrolyzedto the acrylic acid. Suitable hydrolysis conditions are known to thoseskilled in the art and include hydroxide such as lithium hydroxide,sodium hydroxide, potassium hydroxide and cesium hydroxide in thepresence of water. One of ordinary skill will recognize that suchhydrolysis can be performed at temperatures of about 25° C. to about100° C. In some embodiments, the hydrolysis of an acrylate of formula Nis heated to reflux.

In some embodiments, an acrylic acid of formula Q is reacted withHN(R⁴)₂ to form a compound of formula G. In some embodiments, an acrylicacid of formula Q is treated with an amide coupling agent in thepresence of HN(R⁴)₂ to form a compound of formula G. Suitable amidecoupling agents include HOBt, HOAt, HAMDU, HAMTU, PyBOP, PyBrOP, TBTU,HATU and T3P. One of ordinary skill will recognize that the use of suchamide coupling reagents requires the use of a base. Suitable basesinclude organic bases such as triethylamine, diisopropylethyl amine,pyridine, 4-dimethylpyridine (DMAP), and the like.

In some embodiments, a compound of formula Q is reacted with achlorinating agent such as thionyl chloride to form an acyl chloride,which is then reacted with HN(R⁴)₂ to form a compound of formula G.

In some embodiments, the present invention provides a method ofproviding a compound of formula V:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R² and m is as defined above with respect to a compound offormula Z,

comprising the steps of:

(a) providing a compound of formula L:

wherein R² is as defined above for a compound of formula V;

(b) reacting said compound of formula L with

to form a compound of formula R:

wherein R₂ is as defined above for a compound of formula V;

(c) reacting said compound of formula R to provide a compound of formulaJ:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹ and R² is as defined above for a compound of formula V;and

(d) reacting said compound of formula J with a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula V,

in the presence of a sterically-hindered nucleophilic base to provide acompound of formula V.

In some embodiments, the present invention provides a method ofproviding a compound of formula V:

or a pharmaceutically acceptable salt thereof, wherein each of R, R^(x),R^(y), R¹, R² and m is as defined above with respect to a compound offormula Z,

comprising the steps of:

(a) providing a compound of formula L:

wherein R² is as defined above for a compound of formula V;

(b) reacting said compound of formula L with an alcohol having theformula HO—R to form a compound of formula M:

wherein each of R and R² is as defined above for a compound of formulaV,

(c) reacting said compound of formula M to provide a compound of formulaN:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹ and R² is as defined above for a compound of formula V;

(d) hydrolyzing said compound of formula N to form a compound of formulaQ:

wherein each of R¹, R² and LG is as defined above for a compound offormula V;

(e) reacting said compound of formula Q with

to form a compound of formula J:

wherein:

LG is halogen, —OSO₂R or —OSO₂CF₃; and

each of R, R¹ and R² is as defined above for a compound of formula V;and

(f) reacting said compound of formula J with a compound of formula E:

wherein each of R^(x), R^(y) and m is as defined above for a compound offormula V,

in the presence of a sterically-hindered nucleophilic base to provide acompound of formula V.

DEFINITIONS

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75th Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.:Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

Unless specified otherwise within this specification, the nomenclatureused in this specification generally follows the examples and rulesstated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F,and H, Pergamon Press, Oxford, 1979, which is incorporated by referenceherein for its exemplary chemical structure names and rules on namingchemical structures. Optionally, a name of a compound may be generatedusing a chemical naming program: ACD/ChemSketch, Version 5.09/September2001, Advanced Chemistry Development, Inc., Toronto, Canada.

Compounds of the present invention may have asymmetric centers, chiralaxes, and chiral planes (e.g., as described in: E. L. Eliel and S. H.Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, NewYork, 1994, pages 1119-1190), and occur as racemates, racemic mixtures,and as individual diastereomers or enantiomers, with all possibleisomers and mixtures thereof, including optical isomers, being includedin the present invention.

The term “aliphatic” or “aliphatic group,” as used herein, denotes amonovalent hydrocarbon radical that is straight-chain (i.e.,unbranched), branched, or cyclic (including fused, bridged, andspiro-fused polycyclic). An aliphatic group can be saturated or cancontain one or more units of unsaturation, but is not aromatic. Unlessotherwise specified, aliphatic groups contain 1-6 carbon atoms. However,in some embodiments, an aliphatic group contains 1-10 or 2-8 carbonatoms. In some embodiments, aliphatic groups contain 1-4 carbon atomsand, in yet other embodiments, aliphatic groups contain 1-3 carbonatoms. Suitable aliphatic groups include, but are not limited to, linearor branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereofsuch as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “alkyl,” as used herein, means a saturated, straight-chain orbranched aliphatic group. In one aspect, an alkyl group contains 1-10 or2-8 carbon atoms. Alkyl includes, but is not limited to, methyl, ethyl,propyl, iso-propyl, n-butyl, sec-butyl, t-butyl, and the like.

The term “alkenyl,” as used herein, means a straight-chain or branchedaliphatic group having one or more carbon-carbon double bonds (i.e.,—CH═CH—). In one aspect, an alkenyl group has from two to eight carbonatoms, and includes, for example, and without being limited thereto,ethenyl, 1-propenyl, 1-butenyl and the like. The term “alkenyl”encompasses radicals having carbon-carbon double bonds in the “cis” and“trans” or, alternatively, the “E” and “Z” configurations. If an alkenylgroup includes more than one carbon-carbon double bond, eachcarbon-carbon double bond is independently a cis or trans double bond,or a mixture thereof.

The term “alkynyl,” as used herein, means a straight-chain or branchedaliphatic radical having one or more carbon-carbon triple bonds (i.e.,—C≡C—). In one aspect, an alkyl group has from two to eight carbonatoms, and includes, for example, and without being limited thereto,1-propynyl (propargyl), 1-butynyl and the like.

The terms “cycloaliphatic,” “carbocyclyl,” “carbocyclo,” and“carbocyclic,” used alone or as part of a larger moiety, refer to asaturated or partially unsaturated cyclic aliphatic monocyclic orbicyclic ring system, as described herein, having from 3 to 10 members,wherein the aliphatic ring system is optionally substituted as definedabove and described herein. Cycloaliphatic groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl,cyclooctenyl, and cyclooctadienyl. The terms “cycloaliphatic,”“carbocyclyl,” “carbocyclo,” and “carbocyclic” also include aliphaticrings that are fused to one or more aromatic or nonaromatic rings, suchas decahydronaphthyl, tetrahydronaphthyl, decalin, orbicyclo[2.2.2]octane.

The term “cycloalkyl,” as used herein, means a saturated cyclicaliphatic monocyclic or bicyclic ring system having from 3-10 members. Acycloalkyl can be optionally substituted as described herein. In someembodiments, a cycloalkyl has 3-6 carbons.

The term “heterocycloalkyl,” as used herein, means a saturated orunsaturated aliphatic ring system in which at least one carbon atom isreplaced with a heteroatom selected from N, S and O. A heterocycloalkylcan contain one or more rings, which may be attached together in apendent manner or may be fused. In one aspect, a heterocycloalkyl is athree- to seven-membered ring system and includes, for example, andwithout being limited thereto, piperidinyl; piperazinyl, pyrrolidinyl,tetrahydrofuranyl and the like.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon, and includes any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen; and a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

The term “halo” or “halogen,” as used herein, means halogen andincludes, for example, and without being limited thereto, fluoro,chloro, bromo, iodo and the like, in both radioactive andnon-radioactive forms.

The term “haloalkyl,” as used herein, means an aliphatic group which issubstituted with one or more halogen atoms. In some embodiments,haloalkyl refers to a perhalogenated aliphatic group. In someembodiments, haloalkyl refers to an alkyl group which is substitutedwith one or more halogen atoms. Exemplary haloalkyl groups include —CF₃,—CCl₃, —CF₂CH₃, —CH₂CF₃, —CH₂(CF₃)₂, —CF₂(CF₃)₂, and the like.

The term “aryl,” alone or in combination, as used herein, means acarbocyclic aromatic system containing one or more rings, which may beattached together in a pendent manner or may be fused. In particularembodiments, aryl is one, two or three rings. In one aspect, the arylhas five to twelve ring atoms. The term “aryl” encompasses aromaticradicals such as phenyl, naphthyl, tetrahydronaphthyl, indanyl,biphenyl, phenanthryl, anthryl and acenaphthyl. An “aryl” group can have1 to 4 substituents, such as lower alkyl, hydroxyl, halo, haloalkyl,nitro, cyano, alkoxy, lower alkylamino and the like.

The term “heteroaryl,” alone or in combination, as used herein, means anaromatic system wherein at least one carbon atom is replaced by aheteroatom selected from N, S and O. A heteroaryl can contain one ormore rings, which may be attached together in a pendent manner or may befused. In particular embodiments, heteroaryl is one, two or three rings.In one aspect, the heteroaryl has five to twelve ring atoms. The term“heteroaryl” encompasses heteroaromatic groups such as triazolyl,imidazolyl, pyrrolyl, pyrazolyl, tetrazolyl, pyridyl, pyrimidinyl,pyrazinyl, pyridazinyl, indolyl, furyl, benzofuryl, thienyl,benzothienyl, quinolyl, oxazolyl, oxadiazolyl, isoxazolyl, and the like.A “heteroaryl” group can have 1 to 4 substituents, such as lower alkyl,hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino andthe like.

It is understood that substituents and substitution patterns on thecompounds of the invention can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art, as well as thosemethods set forth below. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group can have a suitable substituent at each substitutable position ofthe group and, when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent can be either the same or different at everyposition. Alternatively, an “optionally substituted” group can beunsubstituted.

Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups can be on the samecarbon atom or on different carbon atoms, as long as a stable structureresults. The term “stable,” as used herein, refers to compounds that arenot substantially altered when subjected to conditions to allow fortheir production, detection, and, in certain embodiments, theirrecovery, purification, and use for one or more of the purposesdisclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R^(o), —O—(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o); —(CH₂)₀₋₄Ph, which may besubstituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(o); —CH═CHPh, which may be substituted with R^(o);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(o); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o))C(O)R^(o);—N(R^(o))C(S)R^(o); —(CH₂)₀₋₄N(R^(o))C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o)₂; —(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o);—N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o);—(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R^(o);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(o); —(CH₂)₀₋₄SC(O)R^(o); —(CH₂)₀₋₄C(O)NR^(o)₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —SC(S)SR^(o), —(CH₂)₀₋₄OC(O)NR^(o) ₂;—C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o);—C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o); —(CH₂)₀₋₄S(O)₂R^(o);—(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o); —S(O)₂NR^(o) ₂;—(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂; —N(R^(o))S(O)₂R^(o);—N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o); —P(O)R^(o) ₂; —OP(O)R^(o)₂; —OP(O)(OR^(o))₂; SiR^(o) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(o))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(o), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl monocyclic orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, and sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by takingtwo independent occurrences of R^(o) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. Suitable divalent substituents on asaturated carbon atom of R^(●) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R^(*) ₂))₂₋₃O—,and —S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. Suitable divalent substituents thatare bound to vicinal substitutable carbons of an “optionallysubstituted” group include: —O(CR*₂)₂₋₃O—, wherein each independentoccurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may besubstituted as defined below, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, and —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, and —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmonocyclic or bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

As used herein, “hydrazine equivalent” means a chemical reagent that canbe used to introduce a —N—N— moiety into a molecule. Hydrazineequivalents include hydrazine hydrate as well as protected forms ofhydrazine, such as tert-butyl hydrazine carboxylate.

As used herein, “leaving group” refers to a functional group that isdisplaced from a molecule during a chemical reaction. Leaving groupsinclude halogens, as well sulfonate groups, such as tosylate andmesylate.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of whichare incorporated herein by reference in their entirety. Pharmaceuticallyacceptable salts of the compounds of this invention include saltsderived from suitable inorganic and organic acids and bases that arecompatible with the treatment of patients.

Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable acid addition salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

In some embodiments, exemplary inorganic acids which form suitable saltsinclude, but are not limited thereto, hydrochloric, hydrobromic,sulfuric and phosphoric acid and acid metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate. Illustrativeorganic acids which form suitable salts include the mono-, di- andtricarboxylic acids. Illustrative of such acids are, for example,acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric,malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic,p-toluenesulfonic acid and other sulfonic acids such as methanesulfonicacid and 2-hydroxyethanesulfonic acid. Either the mono- or di-acid saltscan be formed, and such salts can exist in either a hydrated, solvatedor substantially anhydrous form. In general, the acid addition salts ofthese compounds are more soluble in water and various hydrophilicorganic solvents, and generally demonstrate higher melting points incomparison to their free base forms.

In some embodiments, acid addition salts of the compounds of formula Iare most suitably formed from pharmaceutically acceptable acids, andinclude, for example, those formed with inorganic acids, e.g.,hydrochloric, sulfuric or phosphoric acids and organic acids e.g.succinic, maleic, acetic or fumaric acid.

Other non-pharmaceutically acceptable salts, e.g., oxalates can be used,for example, in the isolation of compounds of formula I for laboratoryuse, or for subsequent conversion to a pharmaceutically acceptable acidaddition salt. Also included within the scope of the invention are baseaddition salts (such as sodium, potassium and ammonium salts), solvatesand hydrates of compounds of the invention. The conversion of a givencompound salt to a desired compound salt is achieved by applyingstandard techniques, well known to one skilled in the art.

A “pharmaceutically acceptable basic addition salt” is any non-toxicorganic or inorganic base addition salt of the acid compoundsrepresented by formula I, or any of its intermediates. Illustrativeinorganic bases which form suitable salts include, but are not limitedthereto, lithium, sodium, potassium, calcium, magnesium or bariumhydroxides. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as methylamine,trimethyl amine and picoline or ammonia. The selection of theappropriate salt may be important so that an ester functionality, ifany, elsewhere in the molecule is not hydrolyzed. The selection criteriafor the appropriate salt will be known to one skilled in the art.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds produced bythe replacement of a hydrogen with deuterium or tritium, or of a carbonwith a ¹³C- or ¹⁴C-enriched carbon are within the scope of thisinvention. Such compounds are useful, for example, as analytical tools,as probes in biological assays, or as therapeutic agents in accordancewith the present invention.

The term “stereoisomers” is a general term for all isomers of anindividual molecule that differ only in the orientation of their atomsin space. It includes mirror image isomers (enantiomers), geometric(cis/trans) isomers and isomers of compounds with more than one chiralcenter that are not mirror images of one another (diastereomers).

The term “treat” or “treating” means to alleviate one or more symptoms,to eliminate the causation of one or more symptoms, either on atemporary or permanent basis, or to prevent or delay the onset of one ormore symptoms associated with a disorder or condition.

The term “therapeutically effective amount” means an amount of acompound that is effective in treating or lessening the severity of oneor more symptoms of a disorder or condition.

The term “pharmaceutically acceptable carrier” means a non-toxicsolvent, dispersant, excipient, adjuvant or other material which ismixed with the active ingredient in order to permit the formation of apharmaceutical composition, i.e., a dosage form capable of beingadministered to a patient. One example of such a carrier ispharmaceutically acceptable oil typically used for parenteraladministration. Pharmaceutically acceptable carriers are well known inthe art.

When introducing elements disclosed herein, the articles “a,” “an,”“the,” and “said” are intended to mean that there are one or more of theelements. The terms “comprising,” “having” and “including” are intendedto be open-ended and mean that there may be additional elements otherthan the listed elements.

Formulation and Administration

Pharmaceutically Acceptable Compositions

Another embodiment of the invention is a composition comprising acompound of the invention, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier, adjuvant, orvehicle. The amount of compound in a composition of the invention is anamount that is effective to measurably inhibit CRM1 in a biologicalsample or in a patient. In certain embodiments, a composition of theinvention is formulated for administration to a patient in need of thecomposition. The term “patient,” as used herein, means an animal. Insome embodiments, the animal is a mammal. In certain embodiments, thepatient is a veterinary patient (i.e., a non-human mammal patient). Insome embodiments, the patient is a dog. In other embodiments, thepatient is a human.

The phrase “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Compositions of the present invention may be administered orally,parenterally (including subcutaneous, intramuscular, intravenous andintradermal), by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. In some embodiments,provided compounds or compositions are administrable intravenouslyand/or intraperitoneally.

The term “parenteral,” as used herein, includes subcutaneous,intravenous, intramuscular, intraocular, intravitreal, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intraperitonealintralesional and intracranial injection or infusion techniques.Preferably, the compositions are administered orally, subcutaneously,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example, a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions and solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In some embodiments, aprovided oral formulation is formulated for immediate release orsustained/delayed release. In some embodiments, the composition issuitable for buccal or sublingual administration, including tablets,lozenges and pastilles. A provided compound can also be inmicro-encapsulated form.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. Pharmaceutically acceptable compositions of thisinvention may also be administered topically, especially when the targetof treatment includes areas or organs readily accessible by topicalapplication, including diseases of the eye, the skin, or the lowerintestinal tract. Suitable topical formulations are readily prepared foreach of these areas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For ophthalmic use, pharmaceutically acceptable compositions can beformulated as micronized suspensions or in an ointment such aspetrolatum.

Pharmaceutically acceptable compositions of this invention can also beadministered by nasal aerosol or inhalation.

In some embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for intra-peritoneal administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated and the particular modeof administration. In one embodiment, a composition is formulated sothat a dosage of between 0.01-100 mg/kg body weight/day of the inhibitorcan be administered to a patient receiving the composition. In anotherembodiment, the dosage is from about 0.5 to about 100 mg/kg of bodyweight, or between 1 mg and 1000 mg/dose, every 4 to 120 hours, oraccording to the requirements of the particular drug. Typically, thepharmaceutical compositions of this invention will be administered fromabout 1 to about 6 times per day.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

In some embodiments, the composition further includes one or moreadditional therapeutic or prophylactic agents. When the compositions ofthis invention comprise a combination of a compound of the formulaedescribed herein and one or more additional therapeutic or prophylacticagents, both the compound and the additional agent should be present atdosage levels of between about 1 to 100%, and more preferably betweenabout 5 to 95% of the dosage normally administered in a monotherapyregimen. The additional agents can be administered separately, as partof a multiple dose regimen, from the compounds of this invention.Alternatively, the additional agents can be part of a single dosageform, mixed together with a compound of the invention in a singlecomposition.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention can beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, can be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of CRM1 and are, therefore, useful for treating one or moredisorders associated with activity of CRM1. Thus, in certainembodiments, the present invention provides a method for treating aCRM1-mediated disorder comprising the step of administering to a patientin need thereof a compound of the present invention, or pharmaceuticallyacceptable salt or composition thereof. The compounds and compositionsdescribed herein can also be administered to cells in culture, e.g., invitro or ex vivo, or to a subject, e.g., in vivo, to treat, prevent,and/or diagnose a variety of disorders, including those described hereinbelow.

The activity of a compound utilized in this invention as an inhibitor ofCRM1 may be assayed in vitro, in vivo or in a cell line. Detailedconditions for assaying a compound utilized in this invention as aninhibitor of CRM1 are set forth in the Exemplification.

As used herein, the term “CRM1-mediated disorder or condition” or“disorder or condition associated with CRM1 activity” means any diseaseor other deleterious condition in which CRM1 plays a role. Accordingly,another embodiment of the present invention relates to treating orlessening the severity of one or more diseases in which CRM1 plays arole. In some embodiments, the present invention provides methods oftreating a disease associated with expression or activity of p53, p73,p21, pRB, p27, IκB, NFκB, c-Abl, FOXO proteins, COX-2 in a subjectcomprising administering to the patient a therapeutically effectiveamount of a compound described herein. In another embodiment, thepresent invention relates to a method of treating or lessening theseverity of a disease or condition selected from a proliferativedisorder (e.g., cancer), an inflammatory disorder, an autoimmunedisorder, a viral infection, an ophthalmological disorder or aneurodegenerative disorder, the method comprising administering to apatient in need thereof a compound or composition according to thepresent invention. In a more specific embodiment, the present inventionrelates to a method of treating or lessening the severity of cancer.Specific examples of the above disorders are set forth in detail below.

Cancers treatable by the compounds of this invention include, but arenot limited to, hematologic malignancies (leukemias, lymphomas,myelomas, myelodysplastic and myeloproliferative syndromes) and solidtumors (carcinomas such as prostate, breast, lung, colon, pancreatic,renal, ovarian as well as soft tissue and osteosarcomas, and stromaltumors). Breast cancer (BC) can include, Basal-like Breast Cancer(BLBC), Triple Negative Breast Cancer (TNBC) and breast cancer that isboth BLBC and TNBC. In addition, breast cancer can include invasive ornon-invasive ductal or lobular carcinoma, tubular, medullary, mucinous,papillary, cribriform carcinoma of the breast, male breast cancer,recurrent or metastatic breast cancer, phyllodes tumor of the breast,paget's disease of the nipple.

Inflammatory disorders treatable by the compounds of this inventioninclude, but are not limited to, multiple sclerosis, rheumatoidarthritis, degenerative joint disease, systemic lupus, systemicsclerosis, vasculitis syndromes (small, medium and large vessel),atherosclerosis, inflammatory bowel disease, irritable bowel syndrome,Crohn's disease, mucous colitis, ulcerative colitis, gastritis, sepsis,psoriasis and other dermatological inflammatory disorders (such aseczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma,psoriasis, and dermatosis with acute inflammatory components, pemphigus,pemphigoid, allergic dermatitis), and urticarial syndromes. In someembodiments, the disorder or condition associated with CRM1 activity ismultiple sclerosis, irritable bowel syndrome, rheumatoid arthritis,psoriasis or other dermatological inflammatory disorders.

Viral diseases treatable by the compounds of this invention include, butare not limited to, acute febrile pharyngitis, pharyngoconjunctivalfever, epidemic keratoconjunctivitis, infantile gastroenteritis,Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acutehepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellularcarcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children,tonsillitis and pharyngitis in adults, keratoconjunctivitis), latentHSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2infection, latent HSV-2 infection, aseptic meningitis, infectiousmononucleosis, Cytomegalic inclusion disease, Kaposi's sarcoma,multicentric Castleman disease, primary effusion lymphoma, AIDS,influenza, Reye syndrome, measles, postinfectious encephalomyelitis,mumps, hyperplastic epithelial lesions (e.g., common, flat, plantar andanogenital warts, laryngeal papillomas, epidermodysplasiaverruciformis), cervical carcinoma, squamous cell carcinomas, croup,pneumonia, bronchiolitis, common cold, poliomyelitis, rabies,influenza-like syndrome, severe bronchiolitis with pneumonia, Germanmeasles, congenital rubella, varicella, and herpes zoster. Viraldiseases treatable by the compounds of this invention also includechronic viral infections, including hepatitis B and hepatitis C.

Exemplary ophthalmology disorders include, but are not limited to,macular edema (diabetic and nondiabetic macular edema), age-relatedmacular degeneration (wet and dry forms), aged disciform maculardegeneration, cystoid macular edema, palpebral edema, retina edema,diabetic retinopathy, chorioretinopathy, neovascular maculopathy,neovascular glaucoma, uveitis, iritis, retinal vasculitis,endophthalmitis, panophthalmitis, metastatic ophthalmia, choroiditis,retinal pigment epitheliitis, conjunctivitis, cyclitis, scleritis,episcleritis, optic neuritis, retrobulbar optic neuritis, keratitis,blepharitis, exudative retinal detachment, corneal ulcer, conjunctivalulcer, chronic nummular keratitis, ophthalmic disease associated withhypoxia or ischemia, retinopathy of prematurity, proliferative diabeticretinopathy, polypoidal choroidal vasculopathy, retinal angiomatousproliferation, retinal artery occlusion, retinal vein occlusion, Coats'disease, familial exudative vitreoretinopathy, pulseless disease(Takayasu's disease), Eales disease, antiphospholipid antibody syndrome,leukemic retinopathy, blood hyperviscosity syndrome, macroglobulinemia,interferon-associated retinopathy, hypertensive retinopathy, radiationretinopathy, corneal epithelial stem cell deficiency or cataract.

Neurodegenerative diseases treatable by a compound of the inventioninclude, but are not limited to, Parkinson's, Alzheimer's, andHuntington's, and amyotrophic lateral sclerosis (ALS/Lou Gehrig'sDisease). In some embodiments, the disorder or condition associated withCRM1 activity is ALS.

Compounds and compositions described herein may also be used to treatdisorders of abnormal tissue growth and fibrosis including dilativecardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,pulmonary fibrosis, hepatic fibrosis, glomerulonephritis, and otherrenal disorders.

Compounds and compositions described herein may also be used to treatdisorders related to food intake, such as obesity and hyperphagia. Insome embodiments, the disorder or condition associated with CRM1activity is obesity.

In some embodiments, the disorder or condition associated with CRM1activity is muscular dystrophy, arthritis, for example, osteoarthritisand rheumatoid arthritis, ankylosing spondilitis, traumatic braininjury, spinal cord injury, sepsis, rheumatic disease, canceratherosclerosis, type 1 diabetes, type 2 diabetes, leptospiriosis renaldisease, glaucoma, retinal disease, ageing, headache, pain, complexregional pain syndrome, cardiac hypertrophy, musclewasting, catabolicdisorders, obesity, fetal growth retardation, hypercholesterolemia,heart disease, chronic heart failure, ischemia/reperfusion, stroke,cerebral aneurysm, angina pectoris, pulmonary disease, cystic fibrosis,acid-induced lung injury, pulmonary hypertension, asthma, chronicobstructive pulmonary disease, Sjogren's syndrome, hyaline membranedisease, kidney disease, glomerular disease, alcoholic liver disease,gut diseases, peritoneal endometriosis, skin diseases, nasal sinusitis,mesothelioma, anhidrotic ecodermal dysplasia-ID, behcet's disease,incontinentia pigmenti, tuberculosis, asthma, crohn's disease, colitis,ocular allergy, appendicitis, paget's disease, pancreatitis,periodonitis, endometriosis, inflammatory bowel disease, inflammatorylung disease, silica-induced diseases, sleep apnea, AIDS, HIV-1,autoimmune diseases, antiphospholipid syndrome, lupus, lupus nephritis,familial mediterranean fever, hereditary periodic fever syndrome,psychosocial stress diseases, neuropathological diseases, familialamyloidotic polyneuropathy, inflammatory neuropathy, parkinson'sdisease, multiple sclerosis, alzheimer's disease, amyotropic lateralsclerosis, huntington's disease, cataracts, or hearing loss.

In other embodiments, the disorder or condition associated with CRM1activity is head injury, uveitis, inflammatory pain, allergen inducedasthma, non-allergen induced asthma, glomerular nephritis, ulcerativecolitis, necrotizing enterocolitis, hyperimmunoglobulinemia D withrecurrent fever (HIDS), TNF receptor associated periodic syndrome(TRAPS), cryopyrin-associated periodic syndromes, Muckle-Wells syndrome(urticaria deafness amyloidosis), familial cold urticaria, neonatalonset multisystem inflammatory disease (NOMID), periodic fever, aphthousstomatitis, pharyngitis and adenitis (PFAPA syndrome), Blau syndrome,pyogenic sterile arthritis, pyoderma gangrenosum, acne (PAPA),deficiency of the interleukin-1-receptor antagonist (DIRA), subarachnoidhemorrhage, polycystic kidney disease, transplant, organ transplant,tissue transplant, myelodysplastic syndrome, irritant-inducedinflammation, plant irritant-induced inflammation, poison ivy/urushioloil-induced inflammation, chemical irritant-induced inflammation, beesting-induced inflammation, insect bite-induced inflammation, sunburn,burns, dermatitis, endotoxemia, lung injury, acute respiratory distresssyndrome, alcoholic hepatitis, or kidney injury caused by parasiticinfections.

In another embodiment, a compound or composition described herein may beused to treat or prevent allergies and respiratory disorders, includingasthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygentoxicity, emphysema, chronic bronchitis, acute respiratory distresssyndrome, and any chronic obstructive pulmonary disease (COPD).

Another embodiment of the invention is use of a compound of formula I inthe manufacture of a medicament for the treatment of a disorder orcondition associated with CRM1 activity. In further aspects, the presentinvention provides a use of a compound of formula I for the manufactureof a medicament for the treatment of a disease associated withexpression or activity of p53, p73, p21, pRB, p27, IκB, NFκB, c-Abl,FOXO proteins or COX-2 in a subject. In some embodiments, the presentinvention provides a use of a compound of formula I in the manufactureof a medicament for the treatment of any of cancer and/or neoplasticdisorders, angiogenesis, autoimmune disorders, inflammatory disordersand/or diseases, epigenetics, hormonal disorders and/or diseases, viraldiseases, neurodegenerative disorders and/or diseases and ophthalmologicdisorders.

In some embodiments, the present invention provides a method forinhibiting CRM1 in a biological sample or a patient comprisingcontacting the biological sample with, or administering to the patient,a pharmaceutically acceptable salt of a compound of formula I, orpharmaceutically acceptable composition thereof.

Neoplastic Disorders

A compound or composition described herein can be used to treat aneoplastic disorder. A “neoplastic disorder” is a disease or disordercharacterized by cells that have the capacity for autonomous growth orreplication, e.g., an abnormal state or condition characterized byproliferative cell growth, benign or malignant. Exemplary neoplasticdisorders include: carcinoma, sarcoma (e.g., soft tissue), osteosarcoma,metastatic disorders (e.g., tumors arising from prostate, brain, bone,gastrointestinal, lung, breast, ovarian, cervical, pancreas, kidney,head and neck, and liver origin), hematopoietic neoplastic disorders(e.g., leukemias, lymphomas, myeloma and other malignant plasma celldisorders), and metastatic tumors. In one embodiment, the cancer to betreated is selected from breast, ovarian, cervical, gastrointestinal,prostate, colon, lung, renal, brain, liver, and pancreatic cancer.Treatment with the compound may be in an amount effective to ameliorateat least one symptom of the neoplastic disorder, e.g., reduced cellproliferation, reduced tumor mass, etc.

In one embodiment, the neoplastic disorder is a Basal-like breast cancer(BLBC). BLBCs account for up to 15% of breast cancers (BC) and areusually triple negative breast cancer (TNBC), characterized by lack ofER, progesterone receptor PR, and HER-2 amplification. In a specificembodiment, the breast cancer is TNBC. In addition, mostBRCA1-associated BCs are BLBC and TNBC, expressing basal cytokeratinsand EGFR. BLBC is characterized by an aggressive phenotype, highhistological grade, and poor clinical outcomes with high recurrence andmetastasis rates.

Combination Therapies

In some embodiments, a compound described herein is administeredtogether with an additional “second” therapeutic agent or treatment. Thechoice of second therapeutic agent may be made from any agent that istypically used in a monotherapy to treat the indicated disease orcondition. As used herein, the term “administered together” and relatedterms refers to the simultaneous or sequential administration oftherapeutic agents in accordance with this invention. For example, acompound of the present invention may be administered with anothertherapeutic agent simultaneously or sequentially in separate unit dosageforms or together in a single unit dosage form. Accordingly, the presentinvention provides a single unit dosage form comprising a compound offormula I, an additional therapeutic agent, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

In one embodiment of the invention, in which a second therapeutic agentis administered to a subject, the effective amount of the compound ofthe invention is less than its effective amount would be were the secondtherapeutic agent not administered. In another embodiment, the effectiveamount of the second therapeutic agent is less than its effective amountwould be were the compound of the invention not administered. In thisway, undesired side effects associated with high doses of either agentmay be minimized. Other potential advantages (including, withoutlimitation, improved dosing regimens and/or reduced drug cost) will beapparent to those of skill in the art.

Exemplary additional cancer treatments include, for example:chemotherapy, targeted therapies such as antibody therapies, kinaseinhibitors, immunotherapy, and hormonal therapy, epigenetic therapy,proteosome inhibitors, and anti-angiogenic therapies. Examples of eachof these treatments are provided below.

Examples of chemotherapeutic agents used in cancer therapy include, forexample, antimetabolites (e.g., folic acid, purine, and pyrimidinederivatives) and alkylating agents (e.g., nitrogen mustards,nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes,aziridines, spindle poison, cytotoxic agents, topoisomerase inhibitorsand others). Exemplary agents include aclarubicin, actinomycin,alitretinoin, altretamine, aminopterin, aminolevulinic acid, amrubicin,amsacrine, anagrelide, arsenic trioxide, asparaginase, atrasentan,belotecan, bexarotene, bendamustin, bleomycin, bortezomib, busulfan,camptothecin, capecitabine, carboplatin, carboquone, carmofur,carmustine, celecoxib, chlorambucil, chlormethine, cisplatin,cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, decitabine, demecolcine,docetaxel, doxorubicin, efaproxiral, elesclomol, elsamitrucin,enocitabine, epirubicin, estramustine, etoglucid, etoposide,floxuridine, fludarabine, fluorouracil (5FU), fotemustine, gemcitabine,gliadel implants, hydroxycarbamide, hydroxyurea, idarubicin, ifosfamide,irinotecan, irofulven, ixabepilone, larotaxel, leucovorin, liposomaldoxorubicin, liposomal daunorubicin, lonidamine, lomustine, lucanthone,mannosulfan, masoprocol, melphalan, mercaptopurine, mesna, methotrexate,methyl aminolevulinate, mitobronitol, mitoguazone, mitotane, mitomycin,mitoxantrone, nedaplatin, nimustine, oblimersen, omacetaxine, ortataxel,oxaliplatin, paclitaxel, pegaspargase, pemetrexed, pentostatin,pirarubicin, pixantrone, plicamycin, porfimer sodium, prednimustine,procarbazine, raltitrexed, ranimustine, rubitecan, sapacitabine,semustine, sitimagene ceradenovec, strataplatin, streptozocin,talaporfin, tegafur-uracil, temoporfin, temozolomide, teniposide,tesetaxel, testolactone, tetranitrate, thiotepa, tiazofurine,tioguanine, tipifarnib, topotecan, trabectedin, triaziquone,triethylenemelamine, triplatin, tretinoin, treosulfan, trofosfamide,uramustine, valrubicin, verteporfin, vinblastine, vincristine,vindesine, vinflunine, vinorelbine, vorinostat, zorubicin, and othercytostatic or cytotoxic agents described herein.

Because some drugs work better together than alone, two or more drugsare often given at the same time. Often, two or more chemotherapy agentsare used as combination chemotherapy. In some embodiments, thechemotherapy agents (including combination chemotherapy) can be used incombination with a compound described herein.

Targeted therapy constitutes the use of agents specific for thederegulated proteins of cancer cells. Small molecule targeted therapydrugs are generally inhibitors of enzymatic domains on mutated,overexpressed, or otherwise critical proteins within a cancer cell.Prominent examples are the tyrosine kinase inhibitors such as axitinib,bosutinib, cediranib, desatinib, erolotinib, imatinib, gefitinib,lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, andvandetanib, and also cyclin-dependent kinase inhibitors such asalvocidib and seliciclib. Monoclonal antibody therapy is anotherstrategy in which the therapeutic agent is an antibody whichspecifically binds to a protein on the surface of the cancer cells.Examples include the anti-HER2/neu antibody trastuzumab (Herceptin®)typically used in breast cancer, and the anti-CD20 antibody rituximaband tositumomab typically used in a variety of B-cell malignancies.Other exemplary antibodies include cetuximab, panitumumab, trastuzumab,alemtuzumab, bevacizumab, edrecolomab, and gemtuzumab. Exemplary fusionproteins include aflibercept and denileukin diftitox. In someembodiments, targeted therapy can be used in combination with a compounddescribed herein, e.g., Gleevec (Vignari and Wang 2001).

Targeted therapy can also involve small peptides as “homing devices”which can bind to cell surface receptors or affected extracellularmatrix surrounding a tumor. Radionuclides which are attached to thesepeptides (e.g., RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. An example of such therapy includesBEXXAR®.

Anti-angiogenic therapy can include kinase inhibitors targeting vascularendothelial growth factor (VEGF) such as sunitinib, sorafenib, ormonoclonal antibodies or receptor “decoys” to VEGF or VEGF receptorincluding bevacizumab or VEGF-Trap, or thalidomide or its analogs(lenalidomide, pomalidomide), or agents targeting non-VEGF angiogenictargets such as fibroblast growth factor (FGF), angiopoietins, orangiostatin or endostatin.

Epigenetic therapies include inhibitors of enzymes controllingepigenetic modifications, specifically DNA methyltransferases andhistone deacetylases, which have shown promising anti-tumorigeniceffects for some malignancies, as well as antisense oligonucleotides andsiRNA.

Cancer immunotherapy refers to a diverse set of therapeutic strategiesdesigned to induce the patient's own immune system to fight the tumor.Contemporary methods for generating an immune response against tumorsinclude intravesicular BCG immunotherapy for superficial bladder cancer,prostate cancer vaccine Provenge, and use of interferons and othercytokines to induce an immune response in renal cell carcinoma andmelanoma patients.

Allogeneic hematopoietic stem cell transplantation can be considered aform of immunotherapy, since the donor's immune cells will often attackthe tumor in a graft-versus-tumor effect. In some embodiments, theimmunotherapy agents can be used in combination with a compounddescribed herein.

Hormonal therapy agents include the administration of hormone agonistsor hormone antagonists and include retinoids/retinoic acid, compoundsthat inhibit estrogen or testosterone, as well as administration ofprogestogens.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXEMPLIFICATION Abbreviations

atm Atmosphere

aq. Aqueous

BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

Boc tert-butoxycarbonyl

CDI N,N′-Carbonyldiimidazole

CH₂Cl₂ Dichloromethane

DCC N,N-Dicyclohexylcarbodiimide

DCM Dichloromethane

DBU Diaza(1,3)bicyclo[5.4.0]undecane

DIC N,N′-Diisopropylcarbodiimide

DIPEA N,N-Diisopropylethylamine

DMAP N,N-Dimethyl-4-aminopyridine

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

DPPF Diphenylphosphinoferrocene

EA Ethyl acetate

EDCI N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

eq. equivalent(s)

Et2O Diethylether

EtOAc Ethyl acetate

EtOH Ethanol

EtI Iodoethane

Et Ethyl

Fmoc 9-fluorenylmethyloxycarbonyl

GC Gas chromatography

h hour(s)

HetAr Heteroaryl

HOBt N-Hydroxybenzotriazole

HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

HPLC High performance liquid chromatography

LAH Lithium aluminium hydride

LCMS Liquid Chromatography Mass Spectrometry

MCPBA m-Chloroperbenzoic acid

MeCN Acetonitrile

MeOH Methanol

min Minutes

MeI Iodomethane

MeMgCl Methyl magnesium chloride

Me Methyl

NaOAc Sodium acetate

NMR Nuclear magnetic resonance

NMP N-Methyl pyrrolidinone

o.n. Over night

RT Room Temperature or Retention Time

T3P Propylphosphonic anhydride

TEA Triethylamine

THF Tetrahydrofuran

TLC Thin Layer Chromatography

Throughout the following description of processes it is to be understoodthat, where appropriate, suitable protecting groups will be added to,and subsequently removed from, the various reactants and intermediatesin a manner that will be readily understood by one skilled in the art oforganic synthesis. Conventional procedures for using such protectinggroups, as well as examples of suitable protecting groups, aredescribed, for example, in “Protective Groups in Organic Synthesis”, T.W. Green, P. G. M. Wuts, Wiley-Interscience, New York, (1999). It isalso to be understood that a transformation of a group or substituentinto another group or substituent by chemical manipulation can beconducted on any intermediate or final product on the synthetic pathtoward the final product, in which the possible type of transformationis limited only by inherent incompatibility of other functionalitiescarried by the molecule at that stage to the conditions or reagentsemployed in the transformation. Such inherent incompatibilities, andways to circumvent them by carrying out appropriate transformations andsynthetic steps in a suitable order, will be readily understood to theone skilled in the art of organic synthesis. Examples of transformationsare given below, and it is to be understood that the describedtransformations are not limited only to the generic groups orsubstituents for which the transformations are exemplified. Referencesand descriptions on other suitable transformations are given in“Comprehensive Organic Transformations—A Guide to Functional GroupPreparations” R. C. Larock, VHC Publishers, Inc. (1989). References anddescriptions of other suitable reactions are described in textbooks oforganic chemistry, for example, “Advanced Organic Chemistry”, March, 4thed. McGraw Hill (1992) or, “Organic Synthesis”, Smith, McGraw Hill,(1994). Techniques for purification of intermediates and final productsinclude, for example, normal and reverse-phase chromatography on columnor rotating plate, recrystallization, distillation and liquid-liquid orsolid-liquid extraction, which will be readily understood by the oneskilled in the art. The definitions of substituents and groups are asdescribed for formula I, except where defined differently. The terms“room temperature” and “ambient temperature” shall mean, unlessotherwise specified, a temperature between 16 and 25° C. The term“reflux” shall mean, unless otherwise stated, in reference to a solvent,a temperature at or above the boiling point of the solvent.

Example 1 Synthesis of Intermediate(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid

Synthesis of 3,5-bis(trifluoromethyl)benzothioamide

A 2-L, 3-necked, round-bottomed flask was charged with a solution of3,5-bis(trifluoromethyl)benzonitrile (200 g) in DMF (1 L). The solutionwas then treated with NaSH (123.7 g, 2.0 eq.) and MgCl₂ (186.7 g, 1.0eq.) and the reaction mixture was stirred at RT for 3 hours. The mixturewas poured into an ice-water slurry (10 L) and the compound wasextracted with EtOAc (3×1 L). The combined organic layers were washedwith aqueous saturated brine (3×100 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure to afford 205 g ofdesired crude 3,5-bis(trifluoromethyl)benzothioamide (yield: 90%), whichwas used without purification in the following step.

Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole

A 5-L, 3-necked, round-bottomed flask was charged with a solution of3,5-bis(trifluoromethyl)benzothioamide (205.65 g) in DMF (1.03 L).Hydrazine hydrate (73.2 mL, 2.0 eq.) was added dropwise and the reactionmixture was stirred at RT for 1 h. HCOOH (1.03 L) was added dropwise andthe reaction mixture was refluxed at 90° C. for 3 hours. After beingallowed to cool to RT, the reaction mixture was poured into saturatedaqueous sodium bicarbonate solution (7 L) and extracted with EtOAc (3×1L). The combined organic layers were washed with aqueous saturated brine(3×500 mL), dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure (35° C., 20 mmHg) to afford 180 g of crudecompound. This crude material was stirred with petroleum ether (3×500mL), filtered and dried to obtain 160 g. of3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole obtained as a paleyellow solid (yield: 75%).

Synthesis of (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

A 2-L, 3-necked, round-bottomed flask was charged with a solution of3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (160 g) in DMF (960mL). The solution was treated with DABCO (127.74 g, 2 eq.) and stirredfor 30 min before adding (Z)-isopropyl 3-iodoacrylate (150.32 g, 1.1eq.) dropwise. After ca. 1 hour, the reaction mixture was poured into anice-water slurry (5 L) and extracted with EtOAc (3×1 L). The combinedorganic layers were washed with aqueous saturated brine (3×100 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure (35° C., 20 mmHg) to afford 250 g of crude compound that waspurified by column chromatography (60/120 silica gel) using a ethylacetate/n-hexane gradient (the column was packed in hexane and thedesired compound started eluting from 2% EtOAC/n-hexane). Fractionscontaining the desired compounds were combined to afford 138 g the puredesired compound (yield: 61%).

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid

In a 5-L, 3-necked, round-bottomed flask, (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(130 g, 1.0 eq.) was dissolved in THF (1.3 L). A solution of LiOH (69.3g, 5.0 eq.) in water (1.3 L) was added dropwise to the solution and thereaction mixture was stirred at room temperature for 4 h before beingquenched with 400 mL ice-water slurry and made acidic (pH=2-3) withdilute aqueous HCl. The mixture was extracted with EtOAc (3×1 L) and thecombined organic layers were washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to afford 110 g ofdesired carboxylic acid (yield: 94%) (cis content=90.0%, transcontent=8.2% by LCMS).

Example 2 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide(I-3)

A 50-mL, 3-necked, round-bottomed flask was charged with a suspension of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.200 g) in 1:1 CH2Cl2: AcOEt (25 mL). 2-Hydrazinopyrazine (0.062g) was added at −40° C. followed by T3P (50%) (0.432 g) and DIPEA (0.147g). The reaction mixture was stirred for 30 min at −40° C. before beingconcentrated under reduced pressure (35° C., 20 mmHg). The crude oil waspurified by preparative TLC using 5% MeOH in CH₂Cl₂ as mobile phase(under ammonia atmosphere) to afford 40 mg (yield: 16%) of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 10.53 (s, 1H), 9.59 (s, 1H), 9.14 (s, 1H),8.53 (s, 2H), 8.29 (s, 1H), 8.13 (s, 1H), 8.06-8.07 (m, 1H), 7.92-7.93(d, J=2.8 Hz, 1H), 7.51-7.53 (d, J=10.4 Hz, 1H), 6.07-6.10 (d, J=10.4Hz, 1H); LCMS for C₁₇H₁₂F₆N₇O [M+H]⁺ predicted: 444.31. found: 444.49(RT 2.70 min, purity: 95.78%).

Example 3 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-2-yl)acrylohydrazidehydrochloride (I-4)

A 500-mL, 3-necked, round-bottomed flask was charged with a suspensionof(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (10 g, 1.0 eq.) in 1:1 CH2Cl2:AcOEt (200 mL) 2-Hydrazinopyridine(3.11 g) was added at −40° C. T3P (50% in ethylacetate) (21.75 g) wasadded dropwise followed by DIPEA (7.36 g) and the reaction mixture wasstirred for 30 min at −40° C. before being concentrated under reducedpressure (35° C., 20 mm Hg) to afford a crude brown oil that waspurified by column chromatography (the compound eluted with 1.3% MeOH inCH₂Cl₂). Fractions containing desired compound were combined to afford6.0 g (yield: 48%)(Z)-3-(3-(3,5-bis-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 10.41 (s, 1H), 9.66 (s, 1H), 8.59 (s, 1H),8.53 (s, 2H), 8.28 (s, 1H), 8.06-8.08 (d, J=5.2 Hz, 1H), 7.48-7.53 (m,1H), 7.49-7.52 (d, J=10.4, 1H), 6.71-6.75 (m, 1H), 6.66-6.68 (d, J=8.4Hz, 1H), 6.07-6.09 (d, J=10.4, 1H). LCMS for C₁₈H₁₂F₆N₆O [M+H]⁺predicted: 443.33. found: 443.44 (RT 2.45 min, purity: 100%).

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyridin-2-yl)acrylohydrazidehydrochloride

A 500-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-2-yl)acrylohydrazide(5.5 g) in Et2O (250 mL). The solution was cooled to 5° C., treated withHCl in 1,4-dioxane, allowed to warm to RT and stirred until completion,as shown by TLC analysis (about 1 h). The solids were filtered on aBüchner funnel, washed with Et₂O and dried under vacuum to afford 5.5 g(yield: 92%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-2-yl)acrylohydrazidehydrochloride. ¹H NMR (400 MHz, DMSO-d6) δ, 11.26 (s, 1H), 10.89 (s,1H), 9.55 (s, 1H), 8.52 (s, 2H), 8.28 (s, 1H), 8.03-8.07 (m, 2H),7.62-7.59 (d, J=10.4 Hz, 1H), 7.21-7.24 (m, 1H), 7.05-7.09 (m, 1H),6.16-6.19 (d, J=10.4 Hz, 1H), LCMS for C₁₈H₁₃F₆N₆O [M+H]⁺ 443.33. found443.44 (RT 3.54 min, purity: 99.0%).

Example 4 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(4-hydroxypiperidin-1-yl)prop-2-en-1-one(I-5)

A 50-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.20 g) in CH₂Cl₂ (10 mL). Piperidin-4-ol (0.07 g, 1.2 eq.) wasadded and the solution was cooled to −60° C. for the addition of T3P(propyl phosphonic anhydride) (0.40 mL, 1.2 eq.) and DIPEA (0.19 mL, 2.0eq.). The reaction mixture was stirred for 30 min before being pouredinto water (50 mL) and extracted with CH₂Cl₂ (2×50 mL). The combinedorganic layers were washed with aqueous saturated brine (50 mL), driedover anhydrous MgSO₄, filtered, and concentrated under reduced pressure(25° C., 20 mmHg). Purification by column chromatography using silica60/120 and MeOH:CH₂Cl₂ as mobile phase. (desired compound startedeluting using 3.0% MeOH/CH₂Cl₂) afforded 0.025 g (yield: 10%) of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(4-hydroxypiperidin-1-yl)prop-2-en-1-one.¹H NMR (400 MHz, CDCl₃) δ, 8.75 (s, 1H), 8.58 (s, 2H), 7.93 (s, 1H),7.08-7.11 (d, J=10.4 Hz, 1H), 6.01-6.04 (d, J=10.4 Hz, 1H), 4.02-4.14(m, 1H), 3.98-4.01 (m, 1H), 3.78-3.85 (m, 1H), 3.47-3.52 (s, 1H),3.32-3.38 (s, 1H), 1.96 (s, 1H), 1.83 (s, 1H), 1.27 (s, 1H), 0.90 (s,1H); LCMS for Chemical Formula: C₁₈H₁₇F₆N₄O₂ [M+H]⁺ 435.34. found 435.24(RT 2.408 min, purity: 89.6%).

Example 5 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyrrolidin-1-yl)acrylamide(I-6)

A cold (−40° C.) solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.35 g) in 1:1 CH₂Cl₂:EtOAc (200 mL) was treated with1-aminopyrrolidine HCl (0.134 g). The mixture was then treated with T3P(50% in EtOAc; 0.77 ml, 1.3 eq.) followed by the slow addition of DIPEA(0.51 ml, 3.0 eq.). The reaction mixture was stirred for 30 min at −40°C. before being quenched with ice-water, and extracted with EtOAc (3×20mL). The combined organic layers were washed with aqueous saturatedbrine, dried with anhydrous Na₂SO₄ and concentrated under reducedpressure (35° C., 20 mmHg) to afford 0.275 g of crude solid.Purification by column chromatography on silica gel (60-120 mesh size)using MeOH in CH₂Cl₂ as mobile phase afforded the pure desired(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyrrolidin-1-yl)acrylamide(7.0 mg yield: 1.7%): ¹H NMR (400 MHz, DMSO-d6) δ, 9.49 (s, 1H), 8.95(s, 1H), 8.53 (s, 2H), 8.28 (s, 1H), 7.4-7.38 (d, J=7.6 Hz, 1H),5.87-5.84 (d, J=10.4 Hz, 1H), 2.86-2.81 (m, 4H), 1.74-1.73 (m, 4H); LCMSfor C₁₇H₁₆F₆N₅O [M+H]⁺ 420.33. found 420.13 (RT 7.76 min, purity:92.4%).

Example 6 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(pyridin-2-yl)acrylohydrazide(I-7)

Synthesis of 2-(1-methylhydrazinyl)pyridine

A 25-mL, 3-necked, round-bottomed flask was charged with 2-bromopyridine(0.31 g) and methyl hydrazine (5.09 g, 34.2 eq.) under nitrogenatmosphere and the mixture was stirred and heated to reflux temperatureat 80-85° C. for 1 hr. The reaction mixture was concentrated underreduced pressure (40° C., 20 mmHg) to afford a yellow oil that wastreated with 10% w/v aqueous Na₂CO₃ and extracted with EtOAc. Theorganic layer was washed with aqueous saturated brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure (40°C., 20 mmHg) to afford a yellow oil (0.40 g), which was used as such inthe following step.

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.43 g), 2-(1-methylhydrazinyl)pyridine (0.15 g, 1.0 eq.) in EtOAc(10 mL) T3P (50% in EtOAc; 1.1 g, 1.5 eq.) and DIPEA (0.40 g, 2.5 eq.)were added under nitrogen atmosphere at −60° C. and the progress of thereaction was monitored by TLC (using 10% MeOH:CH₂Cl₂ as mobile phase andvisualization with UV light). The reaction mixture was concentratedunder reduced pressure (25° C., 20 mmHg) to afford 0.65 g of crudesolid. Purification was performed on Combi-Flash Column chromatographyin CH₂Cl₂ and MeOH (desired compound started eluting at 3.3% MeOH inCH₂Cl₂). The fractions containing the desired compound were combined andconcentrated under reduced pressure (35° C., 20 mm Hg) to afford 90.0 mg(yield: 18%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(pyridin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.79 (brs, 1H), 8.57-8.62 (d,2H), 7.92-7.94 (d, J=11.2 Hz, 1H), 7.59-7.64 (m, 1H), 7.19-7.25 (q, 1H),6.75-6.89 (m, 2H), 5.85-5.88 (d, J=10.8 Hz, 1H), 3.46 (d, 3H); LCMS forC₁₉H₁₅F₆N₆O [M+H]⁺ 457.35. found 456.26 (RT 2.52 min, purity: 100.0%).

Example 7 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(pyrazin-2-yl)acrylohydrazide(I-8)

Synthesis of 2-(1-methylhydrazinyl)pyrazine

In a 25-mL, 3-necked, round-bottomed flask, 2-chloropyrazine (0.5 g) wasdissolved in methyl hydrazine (0.5 g, 1.5 eq.) under nitrogen atmosphereat room temperature. Solid K₂CO₃ (0.9 g, 1.5 eq.) was added and thereaction mixture was stirred and heated to reflux at 80-85° C. for 1.0h. The reaction mixture was then allowed to cool to RT and wasconcentrated under reduced pressure (40° C., 20 mmHg) to afford a yellowoily residue that was treated with 10% w/v aqueous Na₂CO₃ and extractedwith EtOAc. The organic extract was washed with brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure (40°C., 20 mmHg) to afford yellow 0.43 g of a yellow oil that was used assuch in the following step.

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.3 g), 2-(1-methylhydrazinyl)pyrazine (0.12 g, 1.1 eq.) andCH₂Cl₂ (10 mL). T3P (50% in EtOAc; 0.38 g, 1.5 eq.) and DIPEA (0.50 g,3.5 eq.) were added under nitrogen atmosphere at −60° C. monitoring theprogress of the reaction by TLC (using 10% MeOH:CH₂Cl₂ as mobile phaseand visualizing under UV light). The reaction mixture was concentratedunder reduced pressure (25° C., 20 mmHg) to afford 0.265 g of solidcrude. Purification using Combi-Flash Column chromatography usingCH₂Cl₂:MeOH as eluent (desired compound started eluting at 1.5% MeOH inCH₂Cl₂) afforded 75.0 mg of pure compound (yield 23%);(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(pyrazin-2-yl)acrylohydrazide:¹H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.40-9.36 (br s, 1H), 8.52(s, 2H), 8.29-8.27 (d, 2H), 8.15 (s, 1H), 7.925-7.92 (d, 1H), 7.56-7.54(d, J=10.4 Hz, 1H), 6.13-6.10 (d, J=10.4 Hz, 1H), 3.43 (d, 3H); LCMS forC₁₈H₁₄F₆N₇O [M+H]⁺ 458.34. found 458.24 (RT 2.83 min; purity: 96.31%).

Example 8 Synthesis(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(3-methylpyridin-2-yl)acrylohydrazide(I-9)

A 50-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) in EtOAc (20 mL). The solution was cooled to −70° C. andwas treated consecutively with 3-methyl-2-(1-methylhydrazinyl)pyridine(0.135 g, 1.0 eq.), T3P (50% in EtOAc; 1.4 mL, 4 eq.) and DIPEA (0.6 mL,6 eq.). The clear reaction mixture was stirred at −60° C. for 4 hr. Theprogress of the reaction was followed by TLC analysis using 2.5% MeOH inCH₂Cl₂ as mobile phase and visualizing under UV. The reaction mixturewas concentrated under reduced pressure (25° C., 20 mm Hg) to afford acrude compound that was purified by column chromatography (60/120 meshSiO2 and eluting with a MeOH:CH₂Cl₂ gradient). The desired compoundstarted eluting with 0.3-0.4% MeOH in dichloromethane Fractionscontaining the desired material were combined to obtain 0.21 g (yield:40%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(3-methylpyridin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ=10.73 (s, 1H), 9.32 (s, 1H), 8.52 (s, 2H),8.45-8.46 (d, J=4.4 Hz, 1H), 8.29 (s, 1H), 7.97-7.99 (d, J=8 Hz, 1H),7.48-7.50 (d, J=10 Hz, 1H), 7.01-7.05 (m, 1H), 5.86-5.88 (d, J=10 Hz,1H), 3.26 (s, 3H); LCMS for C₂₀H₁₄F₉N₆O [M+H]⁺ 525.35. found 525.19 (RT3.31 min, purity 99.40%).

Example 9 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(5-methylpyridin-2-yl)acrylohydrazide(I-10)

A 50-mL, 3-necked, round bottom flask, charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) in EtOAc (10 mL) was treated with2-hydrazinyl-5-methylpyridine (0.97 g, 1.1 eq.). The mixture was cooledto −60° C. and treated with T3P (propyl phosphonic anhydride; 0.85 mL,2.0 eq.) and DIPEA (0.5 mL, 4.0 eq.). The mixture was stirred for 30 minthen poured into water (50 mL) and extracted with CH₂Cl₂ (2×50 mL). Thecombined organic layers were washed with brine (50 mL), dried overanhydrous MgSO₄, filtered, and concentrated under reduced pressure (25°C., 20 mmHg) to afford a crude compound that was purified by columnchromatography (SiO₂, 60/120 mesh, MeOH:CH₂Cl₂ as mobile phase). Thedesired compound started eluting with 2.5% MeOH:CH₂Cl₂. Fractionscontaining the desired compound were combined and concentrated underreduced pressure to afford 0.130 g (yield: 40%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(5-methylpyridin-2-yl)acrylohydrazide.¹H NMR (400 MHz, CDCl₃) δ, 10.38 (s, exchangeable, 1H), 9.65 (s, 1H),8.54 (s, 2H), 8.40 (s, exchangeable, 1H), 8.29 (s, 1H), 7.90 (s, 1H),7.48-7.51 (d, J=10.4 Hz, 1H), 7.33-7.36 (dd, J=2 Hz, J=6 Hz, 1H),6.61-6.63 (d, J=8.4 Hz, 1H), 6.20-6.23 (d, J=10.4 Hz, 1H), 2.15 (s, 3H);LCMS for C₁₉H₁₅F₆N₆O [M+H]⁺ 457.35. found 457.24 (RT 2.61 min, purity:99.13%).

Example 10 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N-(pyridin-3-yl)acrylohydrazide(I-11)

A 50-mL, 3-necked, round bottom flask charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25) in CH₂Cl₂ (12 mL) was treated with3-(1-methylhydrazinyl)pyridine (0.105 g, 1.2 eq.). The mixture wascooled to −60° C. and treated with T3P (propyl phosphonic anhydride;0.50 mL, 1.2 eq.) and DIPEA (0.24 mL, 2.0 eq.) and stirred for 1 h. Theprogress of the reaction was followed by TLC analysis using 10%MeOH:CH₂Cl₂ as mobile phase and visualizing under UV light. The reactionmixture was then poured into water (50 mL) and extracted with CH₂Cl₂(2×50 mL). The combined organic layers were washed with brine (50 mL),dried over anhydrous MgSO₄, filtered, and concentrated under reducedpressure (25° C., 20 mmHg) to afford crude compound which was purifiedby column chromatography (SiO₂, 60/120 mesh, MeOH:CH₂Cl₂ as mobilephase). The desired compound started eluting in 3.0% MeOH:CH₂Cl₂. Thefractions containing the compound were collected and concentrated underreduced pressure to afford 140 mg (yield: 43%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(pyridin-3-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 10.55 (s, 1H), 9.41 (s, 1H), 9.15 (s, 2H),8.58 (s, 1H), 8.53 (s, 1H), 8.29 (s, 1H), 7.51-7.54 (d, J=10.4 Hz, 1H),7.18-7.22 (m, 2H), 6.05-6.07 (d, J=10.4 Hz, 1H), 3.20 (s, 3H); LCMS forC₁₉H₁₅F₆N₆O [M+H]⁺ 457.35. found 457.19 (RT 2.43 min, purity: 83.48%).

Example 11 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(6-chloropyrimidin-4-yl)acrylohydrazide(I-12)

A 25-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.5 g) and 4-chloro-6-hydrazinopyrimidine (0.20 g, 1.0 eq.) inEtOAc (5.0 mL). The mixture was cooled at −40° C. and treated with T3P(2.3 mL, 2.5 eq.) and DIPEA (0.98 mL, 4.0 eq.). TLC analysis (using 5%MeOH—CH₂Cl₂ as eluent) showed that the starting material was consumedafter 30 min. The reaction mixture was then diluted with CH₂Cl₂, washedwith water, dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure (25° C., 20 mmHg) to afford crude material that wassubjected to preparative TLC purification using 5% MeOH—CH₂Cl₂ with asthe mobile phase. This afforded 250 mg (yield: 36.74%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(6-chloropyrimidin-4-yl-)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6), δ=10.59 (br s, exchangeable, 1H), 9.85 (br s,exchangeable, 1H), 9.52 (s, 1H), 8.50 (s, 2H), 8.38 (s, 1H), 8.27 (s,1H), 7.52-7.55 (d, 1H, J=10.4 Hz), 6.69 (s, 1H), 6.05-6.08 (d, 1H,J=10.4 Hz); LCMS: Calculated for C₁₇H₁₁ClF₆N₇O (M+H)⁺ 478.76. found:478.09 (RT 2.79 min, purity: 97.51%).

Example 12 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-3-yl)acrylohydrazide(I-13)

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) and 3-hydrazinopyridine (0.077 g, 1.0 eq.) in EtOAc (10mL). T3P (50% in EtOAc; 0.52 g, 1.2 eq.) and DIPEA (0.27 g, 2.0 eq.)were added under nitrogen atmosphere at −55 to −60° C. The progress ofthe reaction was followed by TLC analysis using 10% MeOH:CH₂Cl₂ asmobile phase and visualization under UV light. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mmHg) to afford 0.475 gof a crude solid. Purification was performed using Combi-Flash Columnchromatography (with MeOH:CH₂Cl₂). The desired compound started elutingat 2.3% MeOH in CH₂Cl₂. The fractions containing the compound werecombined and concentrated under reduced pressure (35° C., 20 mmHg) toafford 20.0 mg (yield: 6%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-3-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.66 (s, 1H), 8.53 (s, 2H),8.28 (s, 1H), 8.24 (s, 1H), 8.13 (s, 1H), 7.93-7.95 (m, 1H), 7.52-7.54(d, J=10.4 Hz, 1H), 7.09-7.15 (m, 2H), 6.04-6.07 (d, J=10.4 Hz, 1H),LCMS for C₁₈H₁₃F₆N₆O [M+H]⁺ 443.33. found 443.19 (RT 2.19 min, purity:99.60%).

Example 13 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(quinoxalin-2-yl)acrylohydrazide(I-14)

Synthesis of 2-hydrazinylquinoxaline

In a 30-mL sealed tube, 2-chloroquinoxaline (1.0 g) was dissolved inethanol (8 mL) and hydrazine hydrate (8 mL) was added under nitrogenatmosphere at room temperature. The mixture was stirred and heated toreflux temperature (80° C.) for 1 hr. The progress of the reaction wasfollowed by TLC analysis using 10% MeOH:CH₂Cl₂ as mobile phase andvisualization under UV light and/or with ninhydrin. The reaction mixturewas concentrated under reduced pressure (40° C., 20 mmHg) to afford 240mg of a white solid, which was used as such in the following step.

A 50-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) and 2-hydrazinylquinoxaline (0.14 g, 1.2 eq.) in EtOAc.T3P (50% in EtOAc; 0.83 mL, 2.0 eq.) and DIPEA (0.5 mL, 4.0 eq.) wereadded under nitrogen atmosphere at −55 to −60° C. and the reactionmixture was stirred for 2 hr before being concentrated under reducedpressure (25° C., 20 mmHg) to afford 0.150 g of crude solid.Purification using Combi-Flash column chromatography (eluting withMeOH:CH₂Cl₂; desired compound started eluting at 5% MeOH in CH₂Cl₂)afforded 60 mg (yield: 20%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(quinoxalin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ=10.851 (s, 1H), 9.89-9.87 (s, 1H), 9.67 (s,1H), 8.49-8.54 (m, 3H), 8.26 (s, 1H), 8.28 (s, 1H), 7.86-7.88 (d, J=8Hz, 1H), 7.45-7.66 (m, 4H), 6.17-6.20 (d, J=10.4 Hz, 1H); LCMS forC₂₁H₁₄F₆N₇O [M+H]⁺ 494.37. found 494.19 (RT 2.88 min, purity: 100%).

Example 14 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(1,1-dioxotetrahydrothiophen-3yl)acrylohydrazide(I-15)

A 50-mL, 3-necked, round-bottomed flask charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.5 g) in EtOAc (20.0 mL) was treated with2-(1,1-dioxotetrahydrothiophen-3-yl)hydrazine (0.3 g, 1.2 eq.). Themixture was cooled to −60° C. and treated simultaneously with T3P (50%in EtOAc; 2.0 mL, 2 eq.) and DIPEA (1 mL, 4 eq.). The reaction mixturewas stirred for 30 min at −60° C. before being concentrated underreduced pressure (35° C., 20 mmHg) to afford 0.60 g of a solid residue.Purification by column chromatography (SiO2; elution with MeOH:CH₂Cl₂;desired compound eluted at 5% MeOH in CH₂Cl₂) afforded 100 mg(yield=15%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(tetrahydrothiophen-1-1-dioxide-3-yl)acrylohydrazide.¹H NMR (400 MHz, CD3OD) δ=9.57 (s, 1H), 8.64 (s, 2H), 8.10 (s, 1H),7.34-7.36 (d, J=10.4 Hz, 1H), 5.89-5.92 (d, J=10.8 Hz, 1H), 4.01 (m,1H), 3.04-3.26 (m, 4H), 2.27-2.34 (m, 2H). LCMS for C₁₇H₁₅F₆N₅O₃S [M+H]⁺484.40. found 483.39 (RT 2.63 min, purity: 66.39%).

Example 15 Synthesis of(Z)—N-(azepan-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide(I-16)

A 500-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.3 g) in CH₂Cl₂:EtOAc (1:1, 200 mL) and the solution was treatedwith azepan-1-amine (0.137 g) at room temperature. The mixture wascooled to −60° C. and treated first with T3P (50% in EtOAc, 0.78 ml) andthen with DIPEA (0.58 mL). The reaction mixture was stirred for 30 minat −60° C. before being quenched with ice-cold water and extracted withEtOAc (3×20 mL). The combined organic extracts were washed with brine,dried over anhydrous Na2SO4, filtered and concentrated under reducedpressure (35° C., 20 mmHg) to afford 0.57 g of solid. Purification bycolumn chromatography (SiO2, MeOH:CH₂Cl₂ as mobile phase; compoundstarted eluting with 0.1% MeOH in CH₂Cl₂) afforded 90 mg (yield: 24%)(Z)—N-(azepan-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide.¹H NMR (400 MHz, DMSO-d6) δ, 9.61 (s, 1H), 9.49 (s, 1H), 9.14 (s, 1H),8.52 (s, 2H), 8.28 (s, 1H), 7.39-7.97 (d, J=10 Hz, 1H), 6.52-6.49 (d,J=10.4 Hz, 1H), 5.86-5.83 (d, J=10.4 Hz, 1H), 3.00-2.97 (m, 4H),1.58-1.54 (m, 8H) LCMS for C₁₉H₁₉F₆N₅O [M+H]⁺ 448.39. found 448.30 at RT3.22 min purity (96.48%).

Example 16 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2,6-dimethylpyrimidin-4-yl)acrylohydrazide(I-17)

A 50-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.20 g.) dissolved in ethyl acetate (15 mL). The solution wascooled to −40° C. and treated with 4-hydrazinyl-2,6-dimethylpyrimidine(0.078 g, 1 eq.). T3P (50% in EtOAc; 0.7 g, 3.0 eq.) and DIPEA (0.367 g,4.0 eq.) were then added simultaneously and the reaction mixture wasstirred for 30 min at −40° C. The reaction mixture was then allowed towarm to room temperature and was concentrated under reduced pressure(35° C., 20 mmHg) to afford 0.340 g of oily crude compound that waspurified by combi-flash using MeOH:CH₂Cl₂ as mobile phase (the desiredcompound was eluted with 7-8% MeOH in CH₂Cl₂) to afford 50 mg (yield:18%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2,6-dimethylpyrimidin-4-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 10.54 (s, 1H), 9.19 (b, 1H), 8.54 (s, 2H),8.30 (s, 1H), 7.52-7.55 (d, J=10.4, 1H), 6.29 (s, 1H), 6.06-6.08 (d,J=10.4, 1H), 2.33 (s, 3H), 2.13 (s, 3H), LCMS for C₁₉H₁₅F₆N₇O [M+H]⁺472.37. found 472.24 (RT 2.88 min, purity: 99.59%).

Example 17 Synthesis of(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide

Synthesis of 3,5-bis(trifluoromethyl)benzothioamide

A 2-L, 3-necked, round-bottomed flask, charged with a solution of3,5-bis(trifluoromethyl)benzonitrile (200 g) in DMF (1 L), was treatedwith NaSH (123.7 g, 2.0 eq.) and MgCl₂ (186.7 g, 1 eq.). The reactionmixture was stirred at RT for 3 h before being poured into an ice-waterslurry (10 L) and was extracted with EtOAc (3×1 L). The combined organicextracts were washed with brine (3×100 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure (25° C., 20 mmHg) toafford 205 g of crude compound (yield: 90%), which was used in thefollowing step without further purification.

Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole

A 5-L, 3-necked, round-bottomed flask, charged with a solution of3,5-bis(trifluoromethyl)benzothioamide (205.65 g) in DMF (1.03 L) wastreated with hydrazine hydrate (73.16 mL, 2.0 eq.) added dropwise. Thereaction mixture was stirred at room temperature for 1 h before beingtreated with HCOOH (1.028 L) added dropwise. The reaction mixture wasrefluxed at 90° C. for 3 h then cooled to room temperature and pouredinto saturated aqueous NaHCO₃ solution (7 L) and extracted with EtOAc(3×1 L). The combined organic layers were washed with brine (3×500 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure (35° C., 20 mmHg) to afford 180 g of a solid. The solid wassuspended in petroleum ether and the suspension was stirred, filteredand dried to afford the desired triazole as a pale yellow solid (160 g,yield: 75%).

Synthesis of (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate and(E)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

A 2-L, 3-necked, round-bottomed flask, charged with a solution of3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (160 g) in DMF(0.96 L, 6V), was treated with DABCO (127.74 g, 2 eq.) and stirred for30 min. (Z)-isopropyl 3-iodoacrylate (150.32 g, 1.1 eq.) was addeddropwise to the above reaction mixture and stirred for 1 h before beingpoured into an ice-water slurry (5 L) and extracted with EtOAc (3×1 L).The combined organic extracts were washed with brine (3×100 mL), driedover anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure(35° C., 20 mmHg) to afford 250 g of crude compound. Purification bycolumn chromatography (SiO2, 60/120 mesh, elution with EtOAc:hexanesgradient; the desired compounds started eluting in 2-2.5% EtOAc inhexanes) afforded pure cis ester (138 g, yield: 61.6%) and pure transester (11.6 g, yield: 5.2%).

Synthesis of(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid

A 500-mL, 3-necked, round-bottomed flask was charged with a solution of(E)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(5.0 g) in THF (50 mL). The solution was treated with a solution of LiOH(2.66 g, 5.0 eq.) in water (50 mL) and the reaction mixture was stirredat room temperature for 4 h. before being diluted with 40 mL water,acidified (pH=2-3) with dilute aqueous HCl and extracted with EtOAc(3×100 mL). The organic extract was washed with brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford 2.75 g of the desired unsaturated carboxylic acid (yield: 61.6%,purity: 99.0% by LCMS).

Synthesis of(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide

To a solution of(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.75 g) in EtOAc (25 mL) and THF (12.5 mL) was added a solution of2-hydrazinopyrazine (0.23 g) in 12 mL THF at room temperature. T3P (50%in ethyl acetate, 1.52 mL) and DIPEA (1.46 mL) were added dropwise andsimultaneously and the reaction mixture was stirred for 30 min at roomtemperature before being quenched with ice-cold water and extracted withEtOAc (3×25 mL). The combined organic layers were washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure (35°C., 20 mmHg), affording 0.698 g of a crude solid. Trituration first withpetroleum ether then with Et₂O afforded 275 mg (yield: 29%)(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyrazin-2-yl)acrylohydrazide,¹H NMR (400 MHz, DMSO-d6) δ, 10.3 (s, 1H), 9.15 (s, 2H), 8.59 (s, 2H),8.30-8.26 (d, J=14.8 Hz, 1H), 8.13 (s, 1H), 8.06-8.07 (m, 1H), 6.98-6.95(d, J=13.4 Hz, 1H); LCMS for C₁₇H₁₂F₆N₇O [M+H]⁺ 443.31. found 444.19 (RT2.625 min, purity: 99.06%).

Example 18 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-4-yl)acrylohydrazidehydrochloride (I-19)

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) and EtOAc (10.0 mL). 4-Hydrazinylpyridine hydrochloride(0.16 g, 1.2 eq.) was added at −40° C. followed by the simultaneousaddition of T3P (50% in EtOAc, 0.85 mL, 2.0 eq.) and DIPEA (0.49 mL, 4.0eq.). The reaction mixture was stirred for 30 min at −40° C. beforebeing concentrated under reduced pressure (35° C., 20 mmHg) to afford0.35 g of crude material. Purification by column chromatography usingMeOH:CH₂Cl₂ as a mobile phase (compound was eluted with 4% MeOH inCH₂Cl₂) afforded 80 mg (yield: 29.85%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-4-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 10.53 (br s, NH exchangeable, 1H), 9.58 (s,1H), 8.88 (br s, NH exchangeable, 1H), 8.84 (s, 2H), 8.29 (s, 1H),8.09-8.11 (d, 2H), 7.52-7.54 (d, J=10.4 Hz, 1H), 6.66-6.69 (m, 2H),6.06-6.10 (d, J=14.4 Hz, H); LCMS for C₁₈H₁₃F₆N₆O [M+H]⁺ 443.33. found443.24 (RT 2.241 min, purity: 90.17%).

A 25-mL, 3-necked, round-bottomed flask was charged with a cold (0° C.)solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-4-yl)acrylohydrazide(0.08 g) in CH₂Cl₂ (5.0 mL) and treated with 4N HCl in dioxane (0.5 mL).The reaction mixture was allowed to warm to room temperature and stirredfor 4 h before being concentrated under reduced pressure (35° C., 20mmHg) to afford 0.05 g (yield: 40.81%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyridin-4-yl)acrylohydrazide-HClsalt. ¹H NMR (400 MHz, DMSO-d6) δ 13.67 (br s, exchangeable, 1H), 10.67(s, exchangeable, 1H), 9.43 (s, 1H), 8.58 (s, 2H), 8.35-8.38 (m, 4H),7.60-7.62 (d, J=10.4 Hz, 1H), 6.92-6.96 (m, 2H), 611-6.13 (d, J=10.4 Hz,1H); LCMS for C₁₈H₁₃F₆N₆O [M+H]⁺ 443.33. found 443.24 (RT 3.00 min,purity: 90.97%).

Example 19 Synthesis of(Z)—N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide(I-20)

Synthesis of 4-benzylpiperazin-1-amine

A 50-mL, 3-necked, round-bottomed flask was charged with conc. HCl andwater, and the solution was cooled at 0-5° C. for the addition of NaNO₂and benzyl piperazine (5.0 g) under a nitrogen atmosphere. The reactionmixture was stirred for 2.5 h at 0-5° C. before being diluted with waterand extracted with EtOAc (3×100 mL). The combined organic extracts weredried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure (40° C., 20 mmHg) to afford 4.40 g a colorless solid.Purification using combi-flash chromatography (elution with 25.5%EtOAc:hexane) afforded 2.0 g of desired compound (yield: 34.3%).

A cold (−70° C.) solution of 1-benzyl-4-nitroso-4-piperizine (0.8 g) inTHF was treated with excess LAH under a nitrogen atmosphere. Thereaction mixture was allowed to warm up to ambient temperature andstirred 1.0 h before being quenched with water and extracted with EtOAc(3×10 mL). The combined organic extracts were dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure (40° C., 20mmHg) to afford 0.70 g 4-benzylpiperazin-1-amine as a colorless solid.

Synthesis of(Z)—N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.220 g, 1.2 eq.), 4-benzylpiperazin-1-amine (0.10 g, 1.0 eq.) andEtOAc (15 ml). T3P (50% in EtOAc 0.99 g, 3.0 eq.) and DIPEA (0.27 mg,4.0 eq.) were added under nitrogen atmosphere to the cold (−60° C.)solution. The progress of the reaction was followed by TLC analysis(SiO₂, 15% MeOH:CH₂Cl₂ as mobile phase, visualization under UV light).The reaction mixture was quenched in water and extracted with ethylacetate (3×15 mL). The combined organic extracts were dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure (25°C., 20 mmHg) to afford 0.35 g of crude solid. Purification onCombi-flash (eluting with 10% MeOH/CH₂Cl₂) afforded 20 mg (yield: 6%)(Z)—N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide.¹H NMR (400 MHz, DMSO-d6) δ 9.44-9.48 (t, 3H), 9.10 (s, 1H), 8.51 (s,2H), 7.23-7.41 (m, 6H), 6.46-6.49 (d, J=10.4 Hz, 1H), 5.83-5.86 (d,J=10.4 Hz, 1H), 3.47 (s, 2H), 2.81 (s, 4H), 2.23-2.33 (d, 2H) LCMS forC₂₄H₂₃F₆N₆O [M+H]⁺ 525.47. found 525.20 (RT 9.87 min, purity: 100%).

Example 20 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(4-ethylpiperazin-1-yl)acrylamide(I-21)

A cold (−40° C.) solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g) in EtOAc (20 mL) was treated with 4-ethylpiperazin-1-amine(0.12 g). T3P (50% in EtOAc, 0.84 mL) and DIPEA (0.24 mL) were addedsimultaneously and the reaction mixture was stirred for 30 min at −40°C. before being quenched with ice-cold water and extracted with EtOAc(3×20 mL). The combined organic extracts were washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure (35° C.,20 mmHg) to afford 0.280 g of crude compound. Purification bycombi-flash chromatography (eluting with 2% MeOH in CH₂Cl₂) followed bypurification on a preparative TLC plate (eluting with 10% MeOH inCH₂Cl₂) afforded 60 mg(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(4-ethylpiperazin-1-yl)acrylamide.¹H NMR (400 MHz, CF3COOD) δ: 10.75 (s, 1H), 8.31-8.29 (d, J=10.2H), 7.98(s, 1H), 7.21-7.23 (d, 1H), 6.08-6.10 (d, 1H), 3.52-3.54 (m, 3H), 3.36(s, 1H), 3.11 (m, 8H), 1.19-1.22 (m, 3H); LCMS for C₁₉H₂₁F₆N₆O [M+H]⁺463.40. found 463.23 (RT 2.43 min, purity: 98.63%).

Example 21 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-morpholinoacrylamide(I-22)

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.250 g), morpholin-4-amine (0.072 g, 1.0 eq.) and EtOAc (10 mL).The solution was cooled to −60° C. and treated with T3P (50% in EtOAc;0.63 mL, 1.5 eq.) and DIPEA (0.24 mL, 2.0 eq.) under a nitrogenatmosphere. The progress of the reaction was followed by TLC analysisusing 10% MeOH:CH₂Cl₂ as mobile phase and visualization under UV light.Upon completion, the reaction mixture was quenched with water andextracted with EtOAc (3×15 mL). The combined organic extracts were driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure(25° C., 20 mmHg) to afford 0.35 g of a crude solid. Purification(Combi-flash, elution with 3% MeOH:CH₂Cl₂) afforded 100 mg (yield: 33%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-morpholinoacrylamide.¹H NMR (400 MHz, DMSO-d6) δ=9.52 (s, NH exchange, 1H), 8.51 (s, 2H),8.28 (s, 1H), 7.38-7.42 (m, 1H), 6.50-6.53 (d, J=10.4 Hz, 1H), 5.84-5.86(d, J=10.4 Hz, 1H), 3.63 (s, 4H), 2.87 (s, 4H); LCMS for C₁₇H₁₆F₆N₅O₂[M+H]⁺ 436.33. found 436.18 (RT 2.64 min, purity: 100%).

Example 22 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrimidin-4-yl)acrylohydrazide(I-23)

Synthesis of 4-hydrazinylpyrimidine

A solution of 2,4-dichloropyrimidine (2.0 g) in EtOH (25 mL) was cooledto 0-20° C. and treated with hydrazine (2.8 mL). The progress of thereaction was followed by TLC using 10% MeOH:CH₂Cl₂ as mobile phase andvisualizing under UV light. The mixture was concentrated under reducedpressure to afford 3.1 g of crude 2-chloro-4-hydrazinyl-pyrimidine(yield=94.8%).

To a solution of 2-chloro-4-hydrazinyl-pyrimidine (200 mg) dissolved inMeOH (10 mL) was added 10% Pd/C (200 mg) and the suspension was stirredunder a hydrogen atmosphere until shown to be complete by TLC analysis(using 10% MeOH:CH₂Cl₂ as mobile phase and visualizing under UV light).The mixture was filtered through Celite® and concentrated under reducedpressure to afford 250 mg of 4-hydrazinylpyrimidine.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrimidin-4-yl)acrylohydrazide

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (250 mg, 1.0 eq.) and EtOAc (20.0 mL). 4-Hydrazinylpyrimidine (231mg, 3 eq.) was added at −60° C. followed by the simultaneous addition ofT3P (50% in EtOAc; 0.84 mL, 2.0 eq.) and DIPEA (0.24 mL, 2.0 eq.). Thereaction mixture was stirred for 30 min at −60° C. before beingconcentrated under reduced pressure (35° C., 20 mm Hg) to afford 0.20 gof a solid. Purification by column chromatography (eluting with 5% MeOHin CH₂Cl₂) afforded 75 mg of material that was purified by preparativeTLC (using MeOH:CH₂Cl₂ as mobile phase) to provide 13 mg (yield=5%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrimidin-4-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ=10.59 (s, 1H), 9.68 (s, NH exchange, 1H),9.47 (s, NH exchange, 1H), 8.53-8.59 (t, 2H), 8.30 (s, 1H), 8.19-8.20(d, 1H), 7.53-7.56 (d, J=11.2 Hz, 1H), 6.66-6.67 (d, 1H), 6.06-6.09 (d,J=10.4 Hz, 1H); LCMS for C₁₇H₁₂F₆N₇O [M+H]⁺ 444.31. found 444.19 (RT2.39 min, purity: 94.97%).

Example 23 Synthesis of(Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide(I-24)

Synthesis of 4-chloro-3,5-bis(trifluoromethyl)benzamide

A solution 4-chloro-3,5-bis(trifluoromethyl)benzonitrile (1.0 g) in DMSO(10 mL) was treated with solid K₂CO₃ (0.55 g, 1.1 eq.) and H2O2 (30%v/v, 1.0 mL). The reaction mixture was stirred at room temperature for 3h before being poured into ice-cold water (20 mL). The precipitate wasfiltered and washed with petroleum ether to afford 1.0 g of crudedesired primary amide (yield: 90%).

Synthesis of 4-chloro-3,5-bis(trifluoromethyl)benzothioamide

To a solution of 4-chloro-3,5-bis(trifluoromethyl)benzamide (1.2 g) intoluene (20 mL) was added Lawesson's reagent (3.32 g, 2.0 eq.). Thereaction mixture was stirred at 90° C. for 8 h before being cooled toroom temperature and filtered. The filtrate was poured into water andextracted with EtOAc (3×100 mL). The combined organic extracts werewashed with brine (3×50 mL), dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure (25° C., 20 mmHg) to afford 2 g ofcrude compound. The crude compound was purified by combi-flashchromatography (eluting with 7% EtOAc:hexane) to afford 1.0 g of desiredcompound (yield: 79%).

Synthesis of3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole

A solution of 4-chloro-3,5-bis(trifluoromethyl)benzothioamide (1 g) inDMF (10 mL) was treated with hydrazine hydrate (0.32 g, 2.0 eq.) and thereaction mixture was stirred at room temperature for 1 h before addingformic acid (3 mL). The reaction mixture was refluxed at 90° C. for 3 hthen cooled to room temperature, poured into aqueous saturated NaHCO₃(slowly, maintaining temperature 25-30° C.) and extracted with EtOAc(3×100 mL). The combined organic extracts were washed with brine (3×50mL), dried over anhydrous Na₂SO₄, filtered, and concentrated underreduced pressure (25° C., 20 mmHg) to afford 1.5 g of crude compound.Purification by column chromatography (eluting with 40% EtOAc in hexane)afforded 0.50 g of desired compound (yield: 36%).

Synthesis of (Z)-isopropyl3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

A solution of3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (2.1 g) inDMF (20 mL) was treated with DABCO (1.5 g, 2 eq.) and the mixture wasstirred for 30 min before adding (Z)-isopropyl 3-iodoacrylate (1.76 g,1.1 eq.). The reaction mixture was stirred at room temperature for 5 hthen poured into ice-cold water (50 mL) and extracted with EtOAc (3×15mL). The combined organic extracts were washed with brine (3×10 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure (25° C., 20 mmHg) to afford 3.0 g of crude compound.Purification by column chromatography using (60/120 mesh SiO₂, elutionwith 1-1.2% MeOH in CH₂Cl₂) afforded desired unsaturated ester (1.33 g,yield: 52%).

Synthesis of(Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid

A 25-mL, 3-necked, round-bottomed flask was charged with a solution of(Z)-isopropyl3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(1.33 g) in 1:1 THF:water (26 mL). The solution was treated with solidLiOH (0.53 g, 4 eq.) and stirred at room temperature for 4 h beforebeing diluted with 400 ml water, acidified to pH=2-3 with dilute aqueousHCl, and extracted with EtOAc (3×100 mL). The combined organic extractswere washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford 0.8 g of crude compound(yield: 66%).

Synthesis of(Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide

In a 50-mL, 3-necked, round-bottomed flask charged with a solution of(Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.8 g) in 1:1 EtOAc:THF (20 mL). The solution was cooled to −70°C. and treated sequentially with 2-hydrazinopyrazine (0.275 g, 1.2 eq.),T3P (50% in EtOAc; 2.5 mL, 2.0 eq.) and DIPEA (1.44 mL, 4.0 eq.), addeddropwise. The clear reaction mixture was stirred at −60° C. for 1 hbefore being concentrated under reduced pressure (25° C., 20 mm Hg) toafford crude compound. Purification by column chromatography using(60/120 mesh SiO₂, elution with 3-4% MeOH in CH₂Cl₂) afforded 0.30 g(yield: 30%)(Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(pyrazin-2-yl)acrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ=10.53 (s, 1H), 9.58 (s, 1H), 9.11 (s, 1H),8.47 (s, 1H), 8.32 (s, 1H), 8.13 (s, 1H), 8.06 (s, 1H), 7.97 (s, 1H),7.52-7.55 (d, J=10.4 Hz, 1H), 6.08-6.11 (d, J=10.4 Hz, 1H); LCMS forC₁₇H₁₁ClF₆N₇O [M+H]⁺ 478.76. found 478.1 (RT 2.64 min, purity: 100%).

Example 24 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-cyclopropylacrylohydrazide(I-25)

A 100-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.50 g) and CH₂Cl₂ (25 mL). DCC (0.29 g, 1.0 eq.) was added andthe mixture was cooled to 0° C. for the sequential addition ofcyclopropylhydrazine hydrochloride (0.15 g, 1.0 eq.) and DIPEA (0.24 mL,1.0 eq.). The reaction mixture was stirred for 1 h before being pouredinto water (50 mL) and extracted with CH₂Cl₂ (2×50 mL). The combinedorganic extracts were washed with brine (50 mL), dried over anhydrousMgSO₄, filtered, and concentrated under reduced pressure (25° C., 20mmHg) to afford crude compound. Purification by combi-flashchromatography (elution with 1.5-2.5% MeOH in CH₂Cl₂) followed byfurther purification on a preparative TLC plate (eluting with 70% EtOAcin hexane) afforded 15 mg (yield: 2.6%)(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-cyclopropylacrylohydrazide.¹H NMR (400 MHz, DMSO-d6) δ, 9.16 (s, 1H), 8.52 (s, 1H), 8.28 (s, 1H),7.23-7.26 (d, J=10.4 Hz, 1H), 6.40-6.43 (d, J=10.4 Hz, 1H), 4.97 (s,1H), 4.63 (s, 1H), 3.18-3.20 (m, 1H), 0.83-0.87 (m, 2H), 0.65-0.69 (m,2H); LCMS for Chemical Formula: C₁₆H₁₄F₆N₅O [M+H]⁺ 406.31. found 406.19(RT 2.74 min, purity: 98.85%).

Example 25 Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyazetidin-1-yl)acrylamide(I-26)

Synthesis of 1-aminoazetidin-3-ol

A cooled (15-20° C.) solution of azetidin-3-ol hydrochloride (2.0 g) inwater (20 ml) was treated with NaOH (0.8 g in 10 mL water) and themixture was stirred at 15-20° C. for 1 h. The reaction mixture was thencooled to 0° C. and treated sequentially with a NaNO₂ solution (1.89 gin 10 mL water) and acetic acid (1.3 mL). After being stirred for 2 h at0-5° C., the reaction mixture was poured into water (20 mL), acidifiedto pH=2-3 with dilute aqueous HCl and extracted with EtOAc (3×25 mL).The combined organic extracts were washed with brine (20 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford 0.26g desired compound, which was used as such in the following step (LCMSpurity: 59.84%).

A solution of 1-nitrosoazetidin-3-ol (0.25 g) in MeOH (15 mL) was cooledto −75° C. and treated with dilute aqueous HCl (1.5 mL). Zinc powder(1.35 g) was then added portionwise and the reaction mixture was stirredat ca. −70° C. for 3 h before being filtered through Celite® andconcentrated under reduced pressure to afford 90 mg1-aminoazetidin-3-ol, which was used as such in the following step.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyazetidin-1-yl)acrylamide

A 50-mL, 3-necked, round-bottomed flask was charged with(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (200 mg) and THF (20.0 mL). The solution was cooled to −60° C. andtreated with a solution of 1-aminoazetidin-3-ol (65 mg, 1.3 eq.) in THF.T3P (50% in EtOAc; 0.67 mL, 2.0 eq.) and DIPEA (0.51 mL, 2.0 eq.) wereadded simultaneously and the reaction mixture was stirred for 30 min at−60° C. before being allowed to warm to room temperature. The reactionmixture was then concentrated under reduced pressure (35° C., 20 mmHg),affording 100 mg of solid. Purification by column chromatography(elution with 3% MeOH in CH₂Cl₂) afforded 20 mg(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyazetidin-1-yl)acrylamide.¹H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 6.38 (s, 1H), 8.52 (s, 2H),8.26 (s, 1H), 7.32-7.35 (d, J=10.8 Hz, 1H), 6.40 (d, exchangeable, 1H),5.78-5.81 (d, J=10.8 Hz, 1H), 4.14-4.15 (d, 1H), 3.82 (m, 2H), 3.71 (m,2H); LCMS for Chemical Formula C₁₆H₁₄F₆N₅O₂ [M+H]⁺ 422.31. found; 422.19(RT 2.46 min, purity: 91.49%).

Examples 26-31

Examples 26-31 describe novel synthetic methods useful in preparation ofcompounds of the invention (e.g., as precursors to compounds of theinvention, such a compounds described by Formula Z above).

Example 26

Synthesis of Isopropyl Propiolate

A 20-L, four-necked, round-bottomed flask, equipped with additionfunnel, thermometer socket and a mechanical stirrer was charged withpropiolic acid (1000 g, 1 equiv.) and IPA (8 L, 8 Vol.). BF₃-etherate(4.54 kg, 2.0 equiv.) was added slowly from an addition funnel at 25° C.over a period of 30 minutes. The temperature of the reaction mixture wasgradually increased up to 90° C. and the reaction mass was maintained atthat temperature for 3 hrs. GC monitoring after 3 hrs showed thecompletion of the reaction. The reaction mixture was cooled to roomtemperature, quenched with 20 L of ice cold DM water and stirred for 30minutes. 10 L of dichloromethane was added to the reaction mixture andthe reaction mass was stirred for another 30 minutes. The organic layerwas separated and the aqueous layer was reextracted with 5 L ofdichloromethane. The combined organic layers was washed with 10 L ofsaturated brine, dried over anhydrous sodium sulphate, and concentratedunder vacuum at 35° C. to 40° C. (product is volatile) to yield theproduct as a brown liquid (1.32 kg, 81.25%). Purity 89.67% (GC); ¹H NMR(300 MHz, CDCl₃) δ: 1.22 (d, 6H, J=6.6 Hz), 2.85 (s, 1H), 4.98-5.05 (m,1H).

Synthesis of (Z)-isopropyl 3-iodoacrylate

A 20-L, four-necked, round-bottomed flask equipped with addition funnel,thermometer socket and mechanical stirrer was charged with isopropylpropiolic ester (1000 g, 1 equiv.) and acetic acid (3.7 L, 3.7 Vol.) at25° C. and the reaction mass was stirred for 10 minutes. Sodium iodide(2.138 Kg, 1.6 Vol.) was added and the reaction mixture was stirred (adark brown colour was observed). The temperature was increased to 110°C. and the reaction was maintained at that temperature for 1.5 hrs. GCmonitoring showed the completion of the reaction after 1.5 hrs. Thereaction mixture was cooled to room temperature, quenched with ice coldDM water (18.75 L, 18.75 V) and stirred for 30 mins. MTBE (5 L) wasadded to the reaction mass and stirred for another 30 minutes. Theorganic layer was separated and the aqueous layer was reextracted withMTBE (5 L). The combined organic layer was washed with NaHCO₃ (2×10 L),NaHSO₃ (2×5 L), saturated brine solution (5.2 L, 5.2 V), dried oversodium sulphate and concentrated under vacuum at 35° C. to yield(Z)-isopropyl 3-iodoacrylate as a brown liquid (1.49 kg, 70%). Purity87.34% (GC); ¹H NMR (300 MHz, CDCl₃) δ: 1.28 (d, 6H, J=6.3 Hz),5.08-5.131 (m, 1H), 6.83 (d, 1H, J=8.7 Hz), 7.38 (d, 1H, J=8.7 Hz).

Synthesis of 3,5-bis(trifluoromethyl)benzothioamide

A 20-L, multi-necked flask equipped with an over-head stirrer, andthermometer socket was charged with bis(trifluoromethyl)benzonitrile(1.25 kg, 1.0 equiv.) and DMF (6.25 L, 5V), and the resulting mixturewas stirred under nitrogen at room temperature (28° C.). The reactionmixture was cooled to 10° C. and 0.775 g NaSH.H₂O (2 equiv.)

was added over a period of 10 mins. After stirring for 15 minutes,MgCl₂.6H₂O (1.169 kg, 1.1 equiv.) was added portionwise over a period of15 minutes and the reaction was stirred for another 35 minutes. Theprogress of the reaction (green-colored solution) was monitored by HPLCwhich showed 99.6% product and 0.03% benzonitrile. The reaction mixturewas cooled to 0-5° C. and 30% dil. HCl (3.75 L) was added dropwise toadjust the pH to 2-3. The resulting mass was extracted with MTBE (5L×1). The layers were separated and 1 L of DM water was added to theaqueous layer, which was reextracted with MTBE (2.5 L×1). The combinedorganic layers were washed with brine (4.5 L×3), dried and concentratedunder vacuum. Hexane was added to the solid obtained, chased and theproduct was isolated as yellow solid (1.400 Kg, 98.0%). Purity: 99.28%(HPLC). ¹H NMR (300 MHz, CDCl₃) δ: 8.27 (s, 1H), 8.53 (s, 2H), 10.0 (s,1H), 10.38 (s, 1H).

Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole

A 20-L, multi-necked flask equipped with an over-head stirrer andthermometer socket was charged with thioamide (1378 g, 1 equiv.) and DMF(6.89 L, 5V), and the mixture was stirred under nitrogen at roomtemperature (28° C.). The reaction mass was cooled to 10° C. andhydrazine hydrate (505.4 g, 2.0 equiv.) was added dropwise over 2 hourswith stirring. The reaction mass was cooled to 0° C. to 5° C. and formicacid was added over a period of 1 hour (6.89 L, 5V) (exotherm wasobserved and the temperature increased to 20° C.). The reaction mixturewas then heated at 95 to 100° C. for another 12 hrs. The progress of thereaction was monitored by HPLC which showed the formation of 99.5%product. The reaction mass was cooled to 35 to 40° C., added to 20.6 Lof pre-cooled DM water (10 to 15° C.) and stirred for 30 minutes. Thereaction mass was extracted with MTBE (8.26 L). The aqueous layer wasagain extracted with MTBE (5.512 L) and the combined organic layers werewashed with 10% sodium bicarbonate (6.89 L, 2V), brine (6.89 L×3), driedwith sodium sulfate and concentrated under vacuum. Dichloromethane (2V)was added to the yellow solid obtained and stirred at 0 to 5° C. for 1hour, which, on filtration, gave the product as a yellow solid (1156 g,82.2%). Purity: 99.7% (HPLC); ¹H NMR (300 MHz, DMSO) δ: 8.15 (s, 1H),8.55 (s, 2H), 8.79 (s, 1H), 14.5 (s, 1H, NH).

Synthesis of (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

A 10-L, four-necked, round-bottomed flask, equipped with additionfunnel, thermometer socket, mechanical stirrer, and stopper was chargedwith 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (600 g, 1.0eq.), DABCO (480 g, 2.0 eq) and DMF (3.0 L). The reaction mixture wasstirred for 30 minutes. After 30 minutes, a solution of iodo ester(1024.8 g, 2.0 eq) in DMF (1200 mL) was added dropwise over a period of1 hour. The progress of the reaction was monitored by HPLC and showed(Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:62.36% and 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole: 15.1%.After 1 hour further, one equivalent of DABCO (258 g) was added and thereaction was maintained for another hour. HPLC analysis showed theconversion as 75.63% (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate and2% 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole. The reactionmixture was quenched with cold DM water (12 L), stirred for 15 minutes,and extracted with ethyl acetate (2×6 L). The combined organic layerswere washed with saturated brine solution (30%, 2×3 L), dried overanhydrous sodium sulfate (100 g) and concentrated. The crude mass (840g) was taken in a 10 L round bottomed flask and methanol (1200 mL) wasadded. The solution was maintained at 0-5° C. and stirred for 30minutes. The obtained solid was filtered and washed with methanol (200mL), which yielded the product as a white solid (550 g, 65.0%). Purity:87.34% (HPLC); ¹H NMR (300 MHz, CDCl₃) δ: 1.30 (d, 6H, J=6.0 Hz), 5.12(m, 1H), 5.73 (d, 1H, J=10.8 Hz), 7.24 (d, 1H, J=10.8 Hz), 7.91 (s, 1H),8.58 (s, 2H), 9.70 (s, 1H). Cis-isomer:Trans-isomer ratio is 83:8.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid

A 5-L, four-necked, round-bottomed flask equipped with addition funnel,thermometer socket, mechanical stirrer and stopper was charged with THF(1.25 L) and (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(125 g, 1 eq.) at room temperature. The reaction mixture was cooled to0° C. To the stirring solution was added ice cold lithium hydroxidesolution (66.58 g in 1.25 L water) over a period of 30 minutes throughan addition funnel. The reaction temperature was slowly raised to 25° C.and the reaction mass was maintained at that temperature for 2 hours.HPLC monitoring showed the following status:(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid: 87.66%;(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid: 9.91%, (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate 2%.The reaction was continued for another 30 minutes and submitted for HPLCmonitoring((Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid: 88.20%;(E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid: 11.03%. After completion of the reaction, the reaction mixture wasquenched with ice cold water (385 mL) and stirred for 30 minutes. The pHwas adjusted to I-2 with dilute hydrochloric acid (30%, 400 mL) and thereaction mass was extracted with ethyl acetate (3×625 mL). The combinedorganic layers were washed with saturated brine solution (30%, 650 mL),dried over anhydrous sodium sulfate (12.5 g) and concentrated underreduced pressure at 30-35° C. Hexane was added to the crude material andstirred for 30 minutes. The obtained solids were filtered through aBuchner funnel and washed with hexane (250 mL). The solid obtained wasdried for 30 minutes under vacuum and at room temperature for 3-4 hours.The product was isolated as a white powder (92.8 g, 84.36%). Purity: 93%(HPLC); ¹H NMR (300 MHz, DMSO-d6) δ: 5.98 (d, 1H, J=10.2 Hz), 7.48 (d,1H, J=10.2 Hz), 8.2 (s, 1H), 8.50-8.54 (m, 2H), 9.39 (s, 1H).

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one

To a 3-L, four-necked, round-bottomed flask equipped with nitrogeninlet, addition funnel, thermometer socket, mechanical stirrer was added(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (100 g, 1.0 eq.) in DCM (1.8 L, 18 V). The reaction mixture wascooled to −10° C. To the cooled solution, were added HOBT (4.4 g, 0.1eq.), EDC.HCl (80.6 g, 1.5 eq.) and 3,3-difluoroazetidine hydrochloride(44 g, 1.2 eq.). To the resulting mixture at −10° C., was added DIPEA(72 mL, 1.5 eq) dropwise over a period of 1.5 hours. The progress of thereaction was monitored by HPLC analysis which showed the completion ofthe reaction at the end of DIPEA addition. The reaction temperature wasslowly raised to 15° C. to 20° C. (˜2 h). The reaction mixture wasquenched with 1 L ice-water slurry. The organic layer was separated andthe aqueous layer was extracted with DCM (400 mL×2). The organic layerwas washed with saturated brine solution (2×500 ml), dried overanhydrous Na₂SO₄ (10 g) and concentrated under reduced pressure (˜35°C.) to afford crude compound. The crude compound thus obtained wasdissolved in 5 vol. of DIPE and stirred at rt for 30 min. and thenfiltered. Crude weight was 100 g (yield=82.39%) [Cis-85.07% by HPLC,Trans-14.36% by HPLC].

The crude compound thus obtained was further purified byrecrystallisation with ethyl acetate according to the followingprocedure. To a 500-mL, four-necked, round-bottomed flask equipped withmechanical stirrer, thermometer socket and stopper was added 100 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one.To this compound at rt was added ethyl acetate (7 volumes) understirring. However, compound was not completely soluble. Hence, theresulting solution was heated to 60° C. to obtain a clear solution andwas then slowly cooled to −30° C. At −30° C., solution was stirred for20 min. and filtered under suction. The compound obtained was driedunder vacuum at 40-45° C. for 3 h-4 hrs to yield the product as a whitesolid. (Cis-98.9% by HPLC);(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one. ¹H NMR (300 MHz, CDCl₃) δ 9.57 (s, 1H),8.56 (s, 2H), 7.90 (s, 1H), 7.18-7.21 (d, J=10.8 Hz, 1H), 5.61-5.65 (d,J=10.8 Hz, 1H), 4.39-4.45 (m, 4H).

Example 27 Synthesis of (Z)-3-iodoacrylic acid

A 250-mL, three-necked, round-bottomed flask equipped with nitrogeninlet was added propiolic acid (7.0 g, 1.0 eq) dissolved in acetic acid(70 mL, 10V) and sodium iodide (29.96 g, 2.0 eq). The reaction mixturewas refluxed at 1000 C for 2-3 h. The progress of the reaction wasfollowed by TLC analysis on silica gel with 10% MeOH:DCM as mobilephase. SM Rf=0.3 and Product Rf=0.5. Reaction mixture was poured intoice water (700 mL) and neutralized with saturated sodium bicarbonatesolution. The reaction mixture was extracted with EtOAc (3×100 mL). Thecombined organic layers were washed with brine solution (3×100 mL),dried over MgSO4, filtered, and concentrated by rotary evaporation (25°C., 20 mmHg) to afford 12.0 g of crude compound which was purified bycolumn chromatography using silica 60/120 using MeOH:DCM as mobilephase. The column (5×10 cm) was packed in DCM and started eluting inMeOH in gradient manner starting with fraction collection (50-mLfractions) from 2% to 5% MeOH in DCM. Compound started eluting with 2%MeOH in DCM. Fractions containing such TLC profile were combined toobtain 8.0 gm of desired compound (yield 40.44%).

Synthesis of (Z)-1-(3,3-difluoroazetidin-1-yl)-3-iodoprop-2-en-1-one

In a 25-mL, three-necked, round-bottomed flask equipped with nitrogeninlet and a rubber septum, (Z)-3-iodoacrylic acid (0.250 g, 1.0 eq.) wasdissolved in DCM (10 mL, 40 V). The reaction mixture was cooled to 0°C., and DIPEA (0.168 g, 1.1 eq), HATU (0.494 g, 1.1 eq) and3,3-difluoroazetidine hydrochloride (0.179 g, 1.1) were added. Thereaction mixture was stirred at 0° C. for 2-3 hr. The progress of thereaction was followed by TLC analysis on silica gel with 40% ethylacetate in hexane. The reaction mixture was filtered and concentrated byrotary evaporation (25° C., 20 mmHg) to afford 0.3 g of crude compoundwhich was purified by column chromatography using silica 60/120 using40% ethyl acetate in hexane as mobile phase. The column (5×10 cm) waspacked in 5% ethyl acetate in hexane and started eluting in ethylacetate in gradient manner starting with fraction collection (50-mLfractions) from 20% to 30% ethyl acetate in hexane. Compound startedeluting with 20% ethyl acetate in hexane. Fractions containing such TLCprofile were combined to obtain 0.18 g of desired compound (yield52.33%). Mass: [M+H]⁺: 273.8.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one

In a 25-mL, three-necked, round-bottomed flask equipped with nitrogeninlet, 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (0.18 g, 1.0eq.) was dissolved in DMF (5.0 mL, 27.0 V), and DABCO (0.143 g, 2.0 eq)and (Z)-1-(3,3-difluoroazetidin-1-yl)-3-iodoprop-2-en-1-one (0.192 g,1.1 eq) were added. The reaction mixture was stirred at RT for 2-3 hr.The progress of the reaction was followed by TLC analysis on silica gelwith 80% ethyl acetate-hexane as mobile phase, SM Rf==0.60 and ProductRf=0.4. Reaction mixture was poured in to ice water (50 mL) andextracted with EtOAc (3×25 mL). The combined organic layers were washedwith brine solution (3×25 mL), dried over MgSO₄, filtered, andconcentrated by rotary evaporation (25° C., 20 mmHg) to afford 0.3 g ofcrude compound which was purified by column chromatography using silica60/120 using ethyl acetate:hexane as mobile phase. The column (5×10 cm)was packed in hexane and started eluting in ethyl acetate in gradientmanner starting with fraction collection (50-mL fractions) from 40% to45% ethyl acetate in hexane. Compound started eluting with 40% ethylacetate in hexane. Fractions containing such TLC profile were combinedto obtain 70 mg of desired compound (yield 25.64%).

Example 28 Synthesis of (Z)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

Synthesis of isopropyl propiolate. To a mixture of propiolic acid (500g, 7.1 moles) in isopropanol (4000 mL) was added BF3 etherate (2015 g,14.2 moles) at 10° C. After stirring for 10 minutes, the reactionmixture was heated to 90° C. and stirred for 2 hours. The completion ofthe reaction was monitored by TLC. The reaction mixture was brought downto 25 to 30° C. and quenched with crushed ice followed by extractionwith dichloromethane. The organic layer was washed with water and thenwith brine solution. Organic layer was dried over sodium sulfate andconcentrated under vacuum to give the isopropyl propiolate (440 g; 55%).Product was confirmed by ¹H NMR.

Synthesis of (Z)-isopropyl 3-iodoacrylate. To a mixture of isopropylpropiolate (350 g, 3.1 moles) in AcOH (1300 mL) was added NaI (930 g,6.2 moles) at 25° C. The reaction mixture was heated to 115° C. andstirred for 1.5 hrs. The reaction mixture was cooled to 25 to 30° C. andquenched with water followed by extraction with MTBE. The organic layerwas washed with saturated bicarbonate, bisulfate and brine solution. Theorganic layer was dried over sodium sulfate and concentrated undervacuum to give the product (Z)-isopropyl 3-iodoacrylate (626 g; 83.5%).Product was confirmed by ¹H NMR.

Synthesis of 3-isopropoxy-5-(trifluoromethyl)benzonitrile

To a mixture of propan-2-ol (102.96 g 1.76 moles) in DMF (3200 mL, 8 V)at 5° C. was added NaH (122 g, 5.08 moles). The mixture was stirred for2 hours. To this mixture 3-fluoro-5-(trifluoromethyl)benzonitrile (400,2.1 moles) was added dropwise. The temperature of the mass was increasedto 25 to 30° C. and maintained at same temperature for 1 hour. Reactionwas monitored by HPLC. After completion, the reaction mixture wasquenched with ice cold water and extracted with ethyl acetate. The ethylacetate layer was washed with brine, dried over sodium sulfate and thenconcentrated under vacuum to give 530 g (2.31 moles; 110%) of3-isopropoxy-5-(trifluoromethyl)benzonitrile, which was taken as such tonext step with no further purification. HPLC purity—96.5% by area (a/a).

Synthesis of 3-isopropoxy-5-(trifluoromethyl)benzothioamide

3-Isopropoxy-5-(trifluoromethyl)benzonitrile (1000 g, 4.3 moles) wasdissolved in DMF (4000 mL) and sodium hydrogensulfide hydrate (636 g;8.6 moles) was added followed by magnesium chloride hexahydrate (960.2g, 4.7 moles). The reaction mixture was stirred for 1 hr at 25 to 30° C.Reaction completion was monitored by TLC using ethyl acetate:hexane(2:8) as the mobile phase. The reaction mixture was quenched in anice-water slurry (250 mL) and the pH was adjusted to 5 by addition of10% aqueous HCl. The reaction mixture was extracted with MTBE and waswashed with 20% brine solution. The organic layer was concentrated undervacuum to give 1136 g (4.3 moles; 100%) of the title compound, which wastaken as such to next step. HPLC purity—97.37% a/a.

Synthesis of3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole

3-Isopropoxy-5-(trifluoromethyl)benzothioamide (646 g; 2.74 moles) wascombined with hydrazine hydrate (140 g; 4.4 moles) and DMF (3200 mL;5V). The mixture was stirred for 30 minutes and cooled to 10° C. To thisreaction mixture was added formic acid (3200 mL) dropwise. Reactionmixture was heated to 90 to 100° C. and maintained for 12 hrs. Afterreaction completion by HPLC, reaction mass was cooled to 25 to 30° C.and quenched with ice-cold water. The mixture was extracted in MTBE. Theorganic layer was washed with brine followed by aqueous sodiumbicarbonate, and concentrated under vacuum. The residue was chased offusing hexane, the resulting residue was slurried at 10° C. for 1 hour.The solid obtained was filtered and dried for 12 hours at 25 to 30° C.to yield 550 g (2.26 moles: 82%) of the product3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole. HPLCpurity—95.24% a/a.

Synthesis of (Z)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

A mixture of 3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole(500 g, 1.8 moles) and DABCO (417.6 g; 3.6 moles) in DMF (1200 mL) wasstirred for 30 minutes. To this mixture was added (Z)-isopropyl3-iodoacrylate (864 g; 3.6 moles) in DMF (1200 mL) slowly at 25 to 30°C. and the reaction mixture was stirred for 1 hour. After 1 hour, DABCO(208 g; 1 eq) was added and the reaction mixture was stirred for 1 hour.HPLC analysis showed3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole 9.59%,(Z)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:73.76%, (E)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:6.66%. The reaction mass was quenched with water, extracted withdichloromethane and concentrated under vacuum to give the crude product.The crude product was chromatographed using ethyl acetate-hexane systemin 60-120 silica gel to give 310 g (0.8 moles; 44%). HPLC purity—99%a/a.

Example 29 Synthesis of (Z)-isopropyl3-(3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

To a solution of3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole (0.50 g)(prepared according to Example 3) in DMF (1.5 mL) was added DABCO (2equiv). The resulting reaction mixture was stirred for 30 min at roomtemperature then (Z)-isopropyl 3-iodoacrylate (2.0 equiv; preparedaccording to Example 3) was added. The resulting mixture was stirred atrt for 3 hrs. The reaction mixture was quenched with ice-cold water, andextracted with ethyl acetate (3 times). Organic layers were separatedand the combined organic layer was dried over anhydrous sodium sulfate.LC-MS and HPLC analysis revealed 62% cis-isomer and 36% trans-isomer. ¹HNMR (400 MHz, CDCl₃) δ: 9.72 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.30(s, 1H), 7.28 (d, J=8.8 Hz, 1H), 5.71-5.73 (d, J=10.8 Hz, 1H), 5.12-5.18(m, 1H), 3.94 (s, 3H), 1.34 (d, 6H): LCMS for C₁₆H₁₆F₃N₃O₃ [M+H]⁺355.31. found 355.92 at 4.317 min (LCMS 99.82%).

Example 30 Synthesis of (Z)-isopropyl3-(3-(2-chloro-6-isopropoxypyridin-4-yl)-1H-1,2,4-triazol-1-yl)acrylate

To 2-chloro-6-isopropoxy-4-(1H-1,2,4-triazol-3-yl)pyridine (0.5 g)(prepared as in Example 3) in 3 mL of DMF, was added DABCO (0.467 g, 2equiv) and the resulting mixture was stirred for 30 min. A solution of(Z)-isopropyl 3-iodoacrylate (0.990 g, 2 equiv) (prepared as in Example3) was added to the reaction mixture, and the resulting mixture wasstirred for 3 h at room temperature. Reaction mixture was worked up asin Example 3, to obtain 53% cis-isomer and 34% trans isomer 34%.

Example 31 Synthesis of (Z)-isopropyl3-(3-(3-(cyclobutylamino)-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate

To N-cyclobutyl-3-(1H-1,2,4-triazol-3-yl)-5-(trifluoromethyl)aniline(0.5 g) (prepared as in Example 3) in 1.5 mL of DMF, was added DABCO(0.188 g) and the resulting mixture was stirred for 30 min. A solutionof (Z)-isopropyl 3-iodoacrylate (0.404 g) (prepared as in Example 3) wasadded to the reaction mixture, and the resulting mixture was stirred for3 h at room temperature. Reaction mixture was worked up as in Example 3,to obtain 44% cis-isomer and 20% trans-isomer.

Example 32 Assays

Exemplary compounds of the invention were tested in parallel withCompounds X-1, X2 and X-3 (depicted in Table 2), in various assays. Theresults are set forth in Table 2 below.

Inhibition of Nuclear Export

The ability of exemplary compounds of the invention to inhibitCRM1-mediated nuclear export was assessed in a RevGFP assay. Rev is aprotein from human immunodeficiency virus type 1 (HIV-1) and contains anuclear export signal (NES) in its C-terminal domain and a nuclearlocalization signal (NLS) in its N-terminal domain. Nuclear export ofRev protein is dependent on the classical NES/CRM1 pathway (Neville etal. 1997). Nuclear accumulation of Rev can be observed in cells treatedwith specific inhibitors of CRM1, such as LMB (Kau et al. 2003).

In this assay, U2OS-RevGFP cells were seeded onto clear-bottomed, black,384-well plates the day before the experiment. Compounds were seriallydiluted 1:2 in DMEM, starting from 40 μM in a separate, 384-well plate,and then transferred onto the cells. The cells were incubated withcompound for about 1 hr before fixation with 3.7% formaldehyde andnuclei staining with Hoechst 33258. The amount of GFP in cell nuclei wasmeasured and the IC₅₀ of each compound was determined (Kau et al. 2003).Compounds of the invention are considered active in the Rev-GFP assayoutlined above if they have an IC₅₀ of less than about 10 μM, with themost preferred compounds having an IC₅₀ of less than about 1 μM. Theresults of the RevGFP assay appear in Table 2.

Cell Proliferation Assay

The CellTiter 96® AQueous One Solution cell proliferation assay(Promega) was used on MM.1S multiple myeloma cell line to study thecytotoxic and cytostatic properties of the compounds. The assay is basedon the cleavage of the tetrazolium salt, MTS, in the presence of anelectron-coupling reagent PES (phenazine ethosulfate). The MTStetrazolium compound is bioreduced by cells into a colored formazanproduct that is soluble in tissue culture medium. This conversion ispresumably accomplished by NADPH or NADH produced by dehydrogenaseenzymes in metabolically active cells. Assays are performed by adding asmall amount of the CellTiter 96® AQueous One solution reagent directlyto culture wells, incubating for 1-4 hours and then recording theabsorbance at 490 nm with a 96-well plate reader. The absorbancerevealed directly correlates to the cell number and their metabolicactivity.

The cells were seeded at 5×10³ to 1.5×10⁴ cells in each well of a96-well plate in 100 μL of fresh culture medium and adherent cells wereallowed to attach overnight. The stock solutions of the compounds werediluted in cell culture medium to obtain eight concentrations of eachdrug, ranging from 1 nM to 30 μM and DMSO at less than 1% v/v was usedas a negative control. The resulting drug solutions were transferredonto the cells. After 72 h of treatment, 20 μl of CellTiter 96® AQueousreagent was added into each well of the 96-well assay plates and theplate was incubated at 37° C. for 1-4 hours in a humidified, 5% CO₂atmosphere. Then the absorbance of each well was recorded at 490 nmusing a 96-well plate reader. In most cases, the assay was performed intriplicate and the results were presented as half maximal inhibitoryconcentration (IC₅₀). Optical density versus compound concentration wasplotted and analyzed using non-linear regression equations (IDBS XLfit)and the IC₅₀ for each compound was calculated.

Pharmacokinetic (PK) Assay and Brain: Plasma Ratio Determination

AUC.

Blood was collected from mice (N=3) to contribute to the total of 10time points (pre-dose, 5 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours,8 hours, 12 hours and 24 hours post dose). Mice were bled on a rotatingbasis, each mouse contributing 3 time points to the blood collection. Atthe designated time points, animals were anaesthetized under isoflurane,and approximately 110 μL of blood per time point was collected viaretro-orbital puncture into pre-cooled K₂EDTA (anti-coagulant) tubes.Blood samples were put on wet ice and centrifuged (2000 g, 5 min at 4°C.) to obtain plasma within 30 minutes of sample collection. All sampleswere stored frozen at approximately −80° C. until analysis. Prior toanalysis, samples were mixed with internal standard (dexamethasone) inacetonitrile, vortexed, centrifuged, and supernatant was injected foranalysis. Concentration of compounds in plasma was determined usingLC-MS-MS instrumentation (API 4000, Triple Quadruple with electrosprayionization; Acuity Ultra Performance Liquid Chromatography column C18,with MeOH and formic acid as organic solvents). AUC values werecalculated using WinNonlin Professional 6.2 software package,non-compartmental pharmacokinetic model NCA200.

Brain to Plasma (B:P) Ratio.

A separate group of mice (N=3) were dosed (PO at 10 mg/kg) and thensacrificed at the time of maximal plasma concentration (estimatedT_(max) at 2 hours post-dose), at which time terminal plasma and braintissue were collected. Following collection, brain tissue was rinsedwith cold saline, dried on filter paper, weighed and snap-frozen byplacing on dry ice. All samples were stored frozen at approximately −80°C. until analysis. At the time of analysis, brain tissue was homogenized(homogenizing solution PBS, pH 7.4), mixed with internal standard(dexamethasone) in acetonitrile, vortexed, centrifuged, and supernatantwas injected for analysis of compound concentration using LC-MS-MSmethodology (API 4000, Triple Quadruple with electrospray ionization;Acuity Ultra Performance Liquid Chromatography column C18, with MeOH andformic acid as organic solvents). Plasma samples were treated with theidentical method (except homogenization step) and the concentration ofcompound in each matrix was calculated based on generated standardcurves. The results of the PK assay and the B:P ratio determination arepresented in Table 2.

TABLE 2 Assay Results for Compounds of Formula I and Comparators Thereto(A = IC₅₀ value of <=1 μM; B = IC₅₀ value from 1-10 μM; C = IC₅₀ valueof >10 μM; NT = not tested). AUC_(Inf) Cmpd. Rev Cytotoxicity (hr · ng/No. Structure Export Assay mL) B:P* X-1**

A A 209^(‡) NT X-2***

A A 68.3^(†) 1.27^(†) X-3

A A 12300 5.0 I-3

A A 10100 0.71 I-4

A A 10800 1.8 I-5

NT A 3850 1.4 I-6

NT A NT NT I-7

A A 12200 1.5 I-8

A A 4600 2.1 I-9

NT A NT NT I-10

NT A 4170 0.77 I-11

NT A NT NT I-12

A A 24900 0.13 I-13

NT A NT NT I-14

NT A NT NT I-15

NT A NT NT I-16

NT A NT NT I-17

NT A NT NT I-18

NT A 7140 0.28 I-19

NT A 4020 0.2 I-20

NT A NT NT I-21

NT A NT NT I-22

NT A NT NT I-23

NT A NT NT I-24

NT A 3350 0.7 I-25

NT A NT NT I-26

NT A NT NT *Dosed in mice at 10 mg/kg po. **Compound 26 from US2009/0275607. ***Compound 44 from US 2009/0275607. ^(‡)AUC_(Inf) valuesfor compound X-1 dosed in mice at 10 mg/kg po were below the limit ofquantitation. Data reported for 5 mg/kg iv. ^(†)Dosed in rats at 10mg/kg po.

The AUC_(Inf) for compound X-1 was below the limit of detection whendosed in mice at 10 mg/kg po. When dosed at 5 mg/kg iv, compound X-1showed minimal exposure, as indicated by the low AUC_(Inf) of 209hr·ng/mL. The brain to plasma ratio for compound X-1 was not determineddue to its negligible exposure levels when dosed po.

The AUC_(Inf) for compound X-2 was calculated to be 68.3 hr·ng/mL whendosed in rats at 10 mg/kg po. Such exposure levels are exceedingly lowwhen compared to compound X-3 and compounds of formula I of the presentinvention. However, compound X-2 exhibits a moderate brain to plasmaratio. The low AUC_(Inf) coupled with a non-negligible brain to plasmaratio suggests that compound X-2 can cross the BBB despite the lowexposure levels. It is believed that Compound X-2 would have asignificantly higher brain to plasma ratio if its AUC_(Inf) wereincreased.

The AUC_(Inf) for compound X-3 was calculated to be 12300 hr·ng/mL whendosed in rats at 10 mg/kg po, indicating good exposure. However,compound X-3 demonstrated a high B:P ratio of 5.0.

The compounds of Formula I are characterized by AUC_(Inf) of greaterthan about 3300 h·ng/mL, in most instances greater than about 3500h·ng/mL, and a relatively low B:P ratio (<2.5). Generally, greaterexposure levels of a therapeutic agent increase the likelihood of brainpenetration. It is therefore surprising and unexpected that compounds offormula I exhibit high AUC_(Inf) levels and relatively low brain toplasma ratios.

In Vivo and In Vitro Activity of Compounds of the Invention AgainstBreast Cancer

Basal-like breast cancers (BLBC) compose up to 15% of breast cancer (BC)and are usually triple negative breast cancer (TNBC) and characterizedby lack of ER, progesterone receptor PR, and HER-2 amplification. Inaddition, most BRCA1-associated BCs are BLBC and TNBC, expressing basalcytokeratins and EGFR. BLBC is characterized by an aggressive phenotype,high histological grade, and poor clinical outcomes with high recurrenceand metastasis rates. Additional therapies are needed. The activity ofthe compounds of the invention, for example, Compound I-3 was assessedin various breast cancer cell lines both in vitro and in vivo.

Inhibition of TNBC (Triple Negative Breast Cancer) Xenograft In Vivo

MDA-MB-468 (ATCC #HTB-132) triple negative breast cancer cells wereobtained from ATCC. These cells were grown in Leibovitz's L-15 mediumsupplemented with 10% fetal calf serum (FCS), 1% penicillin andstreptomycin, and 2 mM L-glutamine. Cells were sub-cultured by dilutionat a ratio of 1:3. Fifty (50) female SCID mice (Charles River Labs),aged 5 to 6 weeks, with a mean pre-treatment body weight of 19.2 gramswere used. SCID mice were inoculated s.c. in the left flank with 5×10⁶MDA-MB-468 cells. When the tumors reached a mean size of between 100 and200 mm³, mice were randomly and prospectively divided into a vehiclecontrol group of ten (10) mice and five treatment groups of eight (8)mice per group. The groups were as follows:

Vehicle (1% Pluronic, 1% PVP in distilled water)

5 FU 50 mg/kg

Compound I-3 5 mg/kg Monday (M), Wednesday (W), Friday (F)

Compound I-3 15 mg/kg, M, W, F

Compound I-3 25 mg/kg M, W, F

Compound I-3 25 mg/kg M, Thursday (Th).

All administrations were via the oral route. Animals were fed withsterile Labdiet® 5053 (pre-sterilized) rodent chow and sterile water wasprovided ad libitum. Tumors were measured once every two days withmicro-calipers, and tumor volume was calculated as(length×width×width)/2. All animals were weighed every day in order toassess differences in weight among treatment groups and monitor wellnessof animals. Any animals exhibiting a loss of greater than 20% ofstarting weight during the course of the study were euthanized. Anyanimals with a tumor over 1500 mm³ in volume were also euthanized.Survival was recorded daily. Dosing solutions were prepared freshly eachday. Compound I-3 was supplied as a lyophilized powder containing 67.8%drug product with the balance made up of Pluronic F-68 and PVP K29/32.This was prepared by dissolving the lyophilized powder at a rate of 6.64mg/90 μL in sterile water, and diluting as necessary in vehicle (1%Pluronic F-68 and 1% PVP K29/32) in sterile water. All dosing solutionsof Compound I-3 were dosed at 0.1 mL/10 g. Statistical differencesbetween treatment groups were determined using Mann-Whitney Rank Sum orANOVA tests with a critical value of 0.05.

On day 33 post inoculation, the tumors were excised. FIG. 1 is a graphof tumor volume as a function of time and shows that Compound I-3displayed efficacy in a dose dependent manner, inhibiting fromapproximately 60% (5 mg/kg Monday, Wednesday, Friday) to nearly 100% oftumor growth (for 25 mg/kg Monday, Thursday regimen) compared withvehicle-treated animals. In addition, Compound I-3 was well tolerated.

Upon excision, the tumors were also stained for the tumor suppressorproteins (TSPs) FOXO3a, IκB, and p27, and nuclear localization of theTSPs was confirmed by immunohistochemistry.

Inhibition of Proliferation and Cytotoxicity in TNBC and Luminal BC CellLines

The CellTiter 96® AQueous One Solution cell proliferation assay(Promega) was used to study the cytotoxic and cytostatic properties ofCompound I-3 in various TNBC and luminal BC cell lines.

The cells were seeded at 5×10³ to 1.5×10⁴ cells (depending on cell type)in each well of a 96-well plate in 100 μL of fresh culture medium andadherent cells were allowed to attach overnight. The stock solutions ofthe compounds were diluted in cell culture medium to obtain eightconcentrations of each drug, ranging from 1 nM to 30 μM and DMSO at lessthan 1% v/v was used as a negative control. The resulting drug solutionswere transferred onto the cells. After 72 h of treatment, 20 μl ofCellTiter 96® AQueous reagent was added into each well of the 96-wellassay plates and the plate was incubated at 37° C. for 1-4 hours in ahumidified, 5% CO₂ atmosphere. Then the absorbance of each well wasrecorded at 490 nm using a 96-well plate reader. In most cases, theassay was performed in triplicate and the results were presented as halfmaximal inhibitory concentration (IC₅₀). Optical density versus compoundconcentration was plotted and analyzed using non-linear regressionequations (Excel Fit) and the IC₅₀ for each cell line against CompoundI-3 was calculated.

The results of the cell proliferation assay are shown in Table 3. Theresults demonstrate the potent cytotoxicity of Compound I-3 on nine offifteen BC cell lines tested. The compound was considered potent in acell line if it had an IC₅₀ value of less than about 1.0 μM. Cell linesin which Compound I-3 had an IC₅₀ value of less than 1.0 μM wereconsidered sensitive cell lines, while cell lines in which Compound I-3had an IC₅₀ value of greater than 1.0 μM were considered resistant celllines. Seven of the nine sensitive cell lines were TNBC. Genomicanalyses on all BC lines indicated that p53, PI3K/AKT and BRCA1 or 2status did not affect cyototoxicity.

TABLE 3 IC₅₀ values for Compound I-3 in various breast cancer celllines. Cell Line Type IC₅₀ (μM) Cell Line Type IC₅₀ (μM) MDA-MB-468 BaB0.01 HCC-1569 BaA 0.96 MDA-MB-231 BaB 0.01 MDA-MB-157 BaB 1.3 DU4475 Lu0.013 HS578T BaB 1.5 BT-549 BaB 0.02 BT-20 BaA 1.5 MCF12A BaB 0.15HCC-202 Lu/HER+ 5.2 MCF10A BaB 0.18 HCC-1428 Lu 10.4 UACC812 Lu 0.59ZR7530 Lu/HER+ 19 HCC-1143 BaA 0.6

Compound I-3 Induces Apoptosis and Inhibits Long-Term BC Growth

The ability of Compound I-3 to induce apoptosis and to inhibit thelong-term growth of selected BC cell lines was assessed.

MDA-MB-468 TNBC, DU4475 and HS578T TNBC cells were exposed toconcentrations of Compound I-3 ranging from 0 to 10 μM for 24 hours.After 24 hours, whole protein cell extracts were run on immunoblots andwere exposed to antibodies against the proteins indicated in FIGS.2A-2C.

FIGS. 2A-2C are images of immunoblots obtained from a few of the mostresistant and most sensitive breast cancer cell lines described above,including MDA-MB-468 TNBC, DU4475 and HS578T TNBC. The study shows thatCompound I-3 induces apoptosis in the sensitive TNBC and luminal BC celllines (MDA-MB-468 and DU4475, respectively) after 24 hours, as indicatedby the decrease in PARP and caspase 3, two apoptosis markers, and theincrease in cleaved PARP and cleaved caspase 3. In contrast, only anegligible increase in cleaved PARP and cleaved caspase 3 was observedwhen a resistant cell line, HS578T, was treated with Compound I-3.

Long-term growth assays were also conducted, in which MDA-MB-468,MDA-MB-231 and HS578T cells were treated with 1 μM Compound I-3 andincubated for 7 (HS578T) or 10 (MDA-MB-468 and MDA-MB-231) days. At theend of the assay, media was removed from the cells and the remainingcells were stained with crystal violet. The study showed that CompoundI-3 inhibited the long-term growth of all three cell lines, includingboth sensitive (MDA-MB-468 and MDA-MB-231) and resistant (HS578T) BCcell lines.

Compound I-3 Increases Nuclear FOXO3a and IκB in TNBC Cell Lines

MDA-MB-468 TNBC Basal A and BT-20 TNBC Basal B cells were exposed toDMSO or 1 μM Compound I-3 for 24 hours and then stained for FOXO3a orIκB with or without DAPI nuclear stain. The stained cells were examinedfor nuclear localization. Following treatment with Compound I-3, bothFOXO3a and IκB were localized in the cell nucleus, while in DMSO-treatedcells, both FOXO3a and IκB were localized in the cytoplasm.

Effect of Compound I-3 on Anti-Apoptosis and Cell Cycle Proteins in TwoTNBC Lines

The effect of increasing concentrations of Compound I-3 on MDA-MB-468and HS578T cells was examined. MDA-MB-468 and HS578T cells were exposedto increasing concentrations of Compound I-3 for 24 hours and totalcellular protein levels of various proteins was probed with antibodiesagainst the proteins indicated in FIG. 3.

FIG. 3 shows that, despite the approximately 100-fold difference in theIC₅₀ of Compound I-3 in the two cell lines after 72 hours (10 nM versus1.5 μM), a reduction in MCL-1 is observed in both cell lines in responseto increasing concentrations of Compound I-3.

The experiments described in Example 32 indicate that inhibition ofCRM1-mediated nuclear export by the compounds of the invention,including Compound I-3, induces nuclear localization and activation oftumor suppressor gene proteins, resulting in selective apoptosis, cancercell cytotoxicity and tumor growth inhibition.

Example 33 Monoclonal-Antibody Induced Arthritis (CAIA)

BalbC mice were randomly assigned to cages on arrival Day (−1) and eachgroup (n=8) was assigned to the treatment groups shown below with thefollowing regimen:

-   -   Vehicle: PO Day 4, 6, 8, 10    -   Dexamethasone: 1 mg/kg IP Days 4, 6, 8, 10    -   Compound I-4: 4 mg/kg PO, Day 4, 6, 8, 10    -   Compound I-4: 7.5 mg/kg PO, Day 4, 6, 8, 10    -   Compound I-4: 15 mg/kg PO, Day 4, 6, 8, 10

The health status of the animals was examined on arrival. Only animalsin good health were acclimatized to laboratory conditions and were usedin the study. Animals were provided ad libitum a commercial rodent dietand free access to drinking water, supplied to each cage viapolyethylene bottles with stainless steel sipper tubes. Automaticallycontrolled environmental conditions were set to maintain temperature at20-24° C. with relative humidity (RH) of 30-70%, a 12:12 hour light:darkcycle and 10-30 air changes/hr in the study room Temperature, RH andlight cycle were monitored daily by the control computer. Animals weregiven a unique animal identification number and on Day 0 of the studyeach animal received a tail vein injection of antibody cocktail (200 uLof 10 mg/mL). The antibody cocktail was supplied by MD Biosciences(Catalog #: CIA-MAB-50). On day 3, post the single mAb administration,all animals were subjected to LPS (200 uL of 0.5 mg/mL) administrationby a single intraperitoneal (IP) injection. LPS was supplied by MDBiosciences (Catalog #: MDLPS.5). Mice were examined for signs ofarthritogenic responses in peripheral joints on day 0. From diseaseonset, arthritogenic response will be examined on study days 3-8, 10,and 12. Arthritis reactions are reported for each paw according to a 0-4scale in ascending order of severity.

Arthritis Score Grade No reaction, normal 0 Mild, but definite rednessand swelling of 1 the ankle/wrist or apparent redness and swellinglimited to individual digits, regardless of the number of affected digitModerate to severe redness and swelling 2 of the ankle/wrist Redness andswelling of the entire 3 paw including digits Maximally inflamed limbwith involvement 4 of multiple joints

Animals found in a moribund condition, animals with broken skin on anarthritic paw, or with a greater than a 20% decrease in body weight andanimals showing severe pain and enduring signs of severe distress werehumanely euthanized. Severe pain or distress was assessed on a case bycase basis by experienced animal technicians. Briefly however,assessments looked for abnormal vocalizations, isolation from otheranimals, unwillingness to use limbs, abnormal response to handling,tremors and posture. Animals were euthanized by CO₂ inhalation followedby cervical dislocation. Evaluation is primarily based on the meanvalues for arthritis scoring and paw thickness measurements. Statisticalanalysis was also be carried out on body weight. Where appropriate,analysis of the data by ANOVA with Tukey post hoc analysis was appliedto determine significance of treatment effects.

As part of this model, animals lose weight quickly for the first 5-8days and slowly start gaining/losing weight depending on the diseaseprogression. I-4 increased the rate of weight gain compared to vehicleor dexamethasone treatment groups. FIG. 4 is a graph of mean body weightversus time for days 0 to 12 in the antibody-induced male BALB/carthritic mice subjected to the model.

In addition, animals subjected to the CAIA model typically begin todisplay signs of arthritis around Day 4 and as the disease progressestotal arthritis scores increase as a function of time. Treatment withCompound I-4 significantly decreased the total score when compared withvehicle and displayed a dose dependent effect. FIG. 5 is a graph of meantotal paw clinical arthritic scores versus time for days 0-12 inantibody-induced male BALB/c arthritic mice subjected to the indicatedtreatment.

Example 34 PMA Induced Psoriasis Model

BALB/c mice were housed in individually ventilated cages in a controlledenvironment (temperature 22±1° C., humidity 70±5%, and 12 h light/12 hdark cycle) in the animal facility. The mice had access to commerciallyavailable feed pellets and UV-treated potable water ad libitum. 4 micewere housed per individually ventilated cage. Each animal in the cagewas identified by a tail. 8 mice per group mice were randomized intodifferent treatment groups according to body weight. Followingrandomization the mean body weight for all groups was equivalent. Studydesign was Group 1: Naïve, 1% DMSO vehicle (10-30 ul, topical oncedaily), Group 2: PMA, 1% DMSO vehicle (10-30 ul, topical once daily),Group 3: PMA, I-4 10 mg/kg in PVP/Pluronics (oral, M-W-F; Day 1-Day3-Day 5-Day 7), Group 4: PMA, 0.1% betamethasone −25 mg (referencestandard) (topical once daily)

4 ug Phorbol 12-myristate 13-acetate (PMA) in 20 uL of acetone wasapplied every day to mouse ears. Starting from Day 2, PMA-induction ofdermal inflammation/psoriasis manifested with increases in clinicaldisease activity index associated with increased thickness of ear,scaling of ear-skin, and folding of ear-skin. The following parameterswere evaluated: (i) the thickness of the ear, (ii) scaling on the skinof ear. This will be based on a scoring index—0, no scaling; 1, mildscaling; 2, moderate scaling; 3, severe scaling. (iii) folding on theskin of the ear. This will be based on a scoring index—0, no folding; 1,mild folding; 2, moderate folding; 3, severe folding, (iv) the weight ofthe ear (on sacrifice day).

FIG. 6 is a bar graph providing scoring for thickness of the ear,scaling of the skin on the ear and folding of the skin of the ear. Theresults show that oral administration of Compound I-4 at 10 mg/kgreduced mean ear thickness in a statistically significant mannercompared to vehicle. Efficacy obtained with I-4 was comparable topositive control betamethasone. In addition, Compound I-4 was welltolerated.

Example 35 Novel Object Recognition

For novel object recognition test, Zucker rats were placed into a testchamber (dimension 26″×18″×18″; L×W×H). Food and water was not bepermitted during the test. The test had 3 phases: a) Familiarisationphase: Rats were singly placed in test chamber and allowed to freelyexplore for 60 min. The distance travelled by the animal during thisphase was recorded using tracking software (AnyMaze system). The purposeof this phase was to familiarise the animals to the test apparatus. Thistest phase was conducted on day 1. b) Sample phase: On day 2, the ratswere singly placed in the test chamber for 3 min and allowed to freelyexplore the test arena which contained 2 identical novel objects (e.gmetal cube, plastic cylinder) positioned at 2 corners of the testchamber. The distance travelled by the animal during this sample phasewas automatically recorded, as well as the time spent by the animalinteracting with the novel objects, using a tracking software system andvisual observation. Interaction with the object was defined as activeinteraction with the animals snout in contact or immediate proximity tothe object. c) Test phase: 1 h after the sample phase, the rats weresingly returned to the test chamber for 3 min and allowed to freelyexplore the test arena which contained 2 objects, one of which was theobject presented during the sample phase, and the second a novel objectwhich was unique to the test phase. The 2 objects were positioned at thesame 2 corners of the test chamber as used for the sample phase. Thedistance travelled by the animal during the test phase was automaticallyrecorded, as well as the time spent by the animal interacting with thenovel and familiar objects, using a tracking software system and visualobservation. Object interaction scores during both the sample and testphase were independently recorded by 2 observers. The final scorerepresents the difference score between each reading. Object preferencescores presented as D1 (i.e time spent exploring novel object−time spentexploring familiar object; therefore positive score represents novelobject preference), and D2 (i.e D1/a+b; D1 score divided by overallobject exploration time).

FIG. 7 provides a set of graphs showing object preference of untreatedand I-4 treated Zucker rats. From FIG. 7 it can be seen that CompoundI-4 orally administered at 0.625, 1.25 and 2.5 mg/kg doses inducedtrends of improved novel object recognition in Zucker rats and I-4 waswell tolerated.

Example 36 Obese Zucker Rats Feeding Study

Male Zucker (fa/fa) rats and male Zucker lean rats (both from CharlesRiver) at 10 weeks of age—a timepoint at which the Zucker fa/fa ratsshould show elevated food intake, body mass and elevated plasma lipidprofile relative to their “lean” counterparts were singly housed inplastic bottomed cages and were given 14 days of habituation. Duringthis period, animal body weight, food and water intakes were recordeddaily. All animals were given ad-lib access to standard lab chow andwater throughout the study. Once the 14 day baseline intake data werecollected, the Zucker obese rats were assigned into treatment groupsbased on equivalent baseline data, i.e. all Zucker obese rats hadequivalent daily food/water intakes and body weights. During this phasethe rats also received two vehicle administrations as familiarisation tothe dosing procedure. Immediately after the baseline phase, thetreatment phase commenced. Test article and vehicle were administered atapproximately 1 h prior to onset of the dark cycle. Dose schedulingvaried according to group: 5× weekly dosing was Monday-Friday The studydesign was the following: Group A=Zucker lean male rats, vehicletreatment 5× week, oral, n=6, Group B=Zucker obese male rats, vehicletreatment 5× week, oral, n=6, Group C=Zucker obese male rats, I-4 2.5mg/kg 5× week, oral, n=6.

Daily body weight, food and water intake were measured at approximatelythe same time of the day. At day −1 and day 7 of treatment phase.

FIG. 8A provides cumulative and average food intake in obese and leanZucker rats (W/O indicates washout period). Oral administration ofCompound I-4 at 2.5 mg/kg 5× weekly reduced mean and cumulative foodintake in obese (fa/fa) Zucker rats. Compound I-4 was well tolerated.

FIG. 8B provides average and percent body weight in obese and leanZucker rats (W/O indicates washout period). Oral administration of I-4at 2.5 mg/kg 5× weekly significantly reduced weight gain compared toZucker fa/fa controls. 2 day washout phase, body weight gain stillreduced compared to Zucker fa/fa controls. I-4 was well tolerated.

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The relevant teachings of all patents, published applications andreferences cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.